Calmodulin inhibitors, chk2 inhibitors and rsk inhibitors for the treatment of ribosomal disorders and ribosomapathies

ABSTRACT

The present invention relates to methods, compositions and kits for treatment of ribosomal disorders and ribosomopathies, e.g. Diamond Blackfan anemia (DBA). In some embodiments, the invention relates to the use of novel classes of compounds, i.e. inhibitors of RSK (p90S6K); inhibitors of p70S6K; and inhibitors of rps6, to treat ribosomal disorders and ribosomopathies. In some embodiments, the invention relates to the use of specific Chk2 inhibitors and to the use of specific phenothiazine derivatives to treat ribosomal disorders and ribosomopathies, e.g. DBA.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation Application of U.S.application Ser. No. 16/315,899 filed on Jan. 7, 2019, which is a 371National Phase Entry of International Patent Application No.PCT/US2017/041851 filed on Jul. 13, 2017 which claims benefit of andpriority to U.S. Provisional Application No. 62/361,631, filed Jul. 13,2016, entitled “CALMODULIN INHIBITORS, CHK2 INHIBITORS AND RSKINHIBITORS FOR THE TREATMENT OF RIBOSOMAL DISORDERS ANDRIBOSOMAPATHIES,” which are hereby incorporated by reference herein intheir entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant nos. HL100001 and HL 134812, awarded by The National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 12, 2021, isnamed 701039-087701USPX_SL.txt and is 27,095 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to methods, compositions andkits for treatment of ribosomal disorders and ribosomopathies, e.g.Diamond Blackfan aanemia (DBA). In some embodiments, the inventionrelates to the use of novel classes of compounds, i.e. inhibitors of RSK(p90S6K); inhibitors of p70S6K; and inhibitors of rps6, to treatribosomal disorders and ribosomopathies. In some embodiments, theinvention relates to the use of specific Chk2 inhibitors as well as tothe use of specific phenothiazine derivatives to treat ribosomaldisorders and ribosomopathies, e.g. DBA.

BACKGROUND OF THE INVENTION

Diamond Blackfan anemia (DBA) is a congenital anemia that presents inchildren, often before one year of age (Vlachos et al., 2008). Theprimary symptom for these patients is a block in erythroiddifferentiation and possible defect in hematopoietic stem cells (HSCs),and some patients also have craniofacial anomalies. Ribosomal proteinS19 (RPS19) was the first gene found mutated in DBA patients(Draptchinskaia et al., 1999). Sequencing of patient samples hasidentified mutations of either large (60s) or small (40s) subunitribosomal proteins in over 50% of patients (Vlachos et al., 2010), mostrecently rps29. Patients are heterozygous for these mutations, alwaysmaintaining a wildtype copy of the affected ribosomal protein gene.

Ribosomal protein knockdown leads to an increase of free ribosomalproteins. Some ribosomal proteins, including RPL11 and RPL5, can preventp53 degradation, as they are able to bind MDM2 and sequester it from p53(Fumagalli et al, 2009). RPL26 has been shown to increase p53 protein byan alternative mechanism, as it can bind p53 mRNA, increasing itstranslation (Tagaki et al., 2005). p53 activation plays an importantrole in DBA pathogenesis, as well as in other diseases where ribosomaland related genes are mutated, now termed ribosomopathies. These include5q-myelodysplastic syndrome, where one copy of RPS14 is lost. p53activation is also a common feature in bone marrow failure disorders,such as Fanconi Anemia (Ceccaldi et al., 2012). In human CD34+ cells,RPS19 knockdown leads to p53 activation (Ebert et al., 2005; Flygare etal., 2005), with increased accumulation in erythroid cells.Differentiation defects can be rescued by p53 inhibition (Dutt et al.,2011). Mouse models of RPS19 mutation or knockdown have hematopoieticdefects that can be rescued by p53 mutation (McGowan et al., 2008; Jaakoet al., 2011). Rps19 has been targeted by morpholino in zebrafishembryos, and the hematopoietic defects in rp111 mutant zebrafish arerescued by p53 knockdown (Danilova et al., 2008; Torihara et al., 2011;Danilova et al., 2011).

Ribosomal protein mutations are common in patients with Diamond Blackfananemia (DBA), who have red cell aplasia and craniofacial abnormalities.The inventors have previously characterized zebrafish mutant rps29, aribosomal protein in the small subunit, that have hematopoietic andendothelial defects (Taylor et al., 2012). Rps29−/− embryos havemorphological defects in the head, as well as decreased hematopoieticstem cells, hemoglobin, and staining of endothelial markers. Consistentwith other models of DBA, knockdown of p53 near completely rescues therps29 mutant phenotype.

The inventors have previously demonstrated that Rps29−/− embryos have adefect in arterial specification, leading to decreased HSCs anddecreased flk1 expression in the intersegmental vessels at 24 hours postfertilization (hpf). Primitive erythropoiesis is also affected, asrps29−/− embryos have less hemoglobin. These embryos also have increasedapoptosis, particularly in the head, and die by five days postfertilization (dpf). p53 pathways are activated in the embryo, and p53mutation rescues all hematopoietic and apoptotic phenotypes. Using thismodel system the inventors discovered that Calmodulin (CAM) inhibitorsand Ca2⁺ inhibitors can rescue the Rps29−/− phenotype and can be used totreat ribosomal disorders or ribosomopathy, e.g. Diamond Blackfan anemia(DBA) (See e.g. US publication 2015/0265627).

While significant progress is being made in the identification ofcompounds for treatment, current treatment options for treatment ofribosomal disorders or ribosomopathy, e.g. a mutation in a ribosomalprotein, are far from optimal, especially for DBA. As such, it is stillimperative to discover novel, effective, and targeted therapies forthese diseases associated with a ribosomal disorder or ribosomopathy,e.g., a mutation in a ribosomal protein. In particular, there is astrong need in the art for improved methods for treatment of DBA withsmall-molecule drugs.

SUMMARY OF THE INVENTION

The present invention is generally directed to methods, compositions andkits for treatment of ribosomal disorders and ribosomapathies, e.g.Diamond Blackfan anemia (DBA). In some embodiments, the inventionrelates to the use of novel classes of compounds, i.e. inhibitors of RSK(p90S6K), e.g. SL and SK; and inhibitors of p70S6K, e.g. PF; andinhibitors of rps6, to treat ribosomal disorders and ribosomopathies. Insome embodiments, the invention relates to the use of specific Chk2inhibitors, e.g. CCT and III, for treatment of ribosomal disorders andribosomapathies, e.g. DBA. In some embodiments, the invention relates tothe use of specific phenothiazine derivatives, e.g. Perphenazine(PerSucc), or ACV, or 221E, or DB-4-088-2 (088-2), or DB-4-088-3(088-3), or DB-4-086 (086), or DB-4-087-2 (087-2), or DB-4-087-3(087-3), or DB-4-089 (089)) to treat ribosomal disorders andribosomopathies, e.g. DBA.

In particular, the present invention is based, in part, upon thediscovery that RSK signaling is upregulated in ribosomal proteindeficient cells, e.g. RPA19 deficient cells. The inventors havediscovered that RSK is activated upon RPS19 deficiency in CD34⁺ cellsand that inhibitors of RSK(p90s6K) as well as inhibitors of p70s6Kincrease hemoglobin (Hb) in rps29−/− zebrafish embryos, an in vivo modelof ribosomal protein defect. The inventors have further determined thatspecific not previously disclosed inhibitors of Chk2 rescue the Hb inrps29−/− embryos, i.e. CCT and III.

In addition, the inventors have identified specific, not previouslydisclosed, phenothiazine derivatives such as ACV; 22E1; PerSucc;DB-4-088-2 (088-2); DB-4-088-3 (088-3); and DB-4-089 (089), which workparticularly well at rescuing morphological defects and hematopoieticand endothelial defects in rps29 −/− zebrafish embryos. Phenothiazinederivative DB-4-088-2 (088-2) advantageously did not change behavior ofzebrafish in a behavior screen, and phenothiazine derivative DB-4-089increased Hb at low concentrations as well as increased eyrthroiddifferentiation. The phenothiazine derivative compounds DB-4-083 (083);DB-4-084 (084); and DB-4-086 (086) did not increase Hb at the same lowconcetrations as DB-4-089.

In certain embodiments, the phenothiazine compound to treat ribosomaldisorders and ribosomopathies, e.g. DBA, is a compound of Formula (I):

wherein:

-   -   X is O or S;    -   R¹ is H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyls, acyl,        aryl, heteroaryl, alkylheteroaryl or alkylaryl;    -   each R is independently H, halo, alkyl, alkyl, alkenyl, alkynyl,        haloalkyl, CN, OH, NH₂, alkylamino, dialkylamino, CO₂H, acyl,        SH, thioalkoxy, SO₂H, or SO₃H; and    -   isomers and pharmaceutically acceptable salts thereof. See        section herein entitled phenothiazine compounds.

In some embodiments, the phenothiazine compound to treat ribosomaldisorders and ribosomopathies, e.g. DBA, is a compound of Formula II:

wherein:

-   -   each R²¹ is independently selected from the group consisting of        H, halo, alkyl, haloalkyl, CN, OH, NH₂, alkylamino,        dialkylamino, CO₂H, acyl, SH, thioalkoxy, SO₂H, and SO₃H;        isomers and pharmaceutically acceptable salts thereof. See        section herein entitled phenothiazine compounds.

In one embodiment, the phenothiazine compound to treat ribosomaldisorders and ribosomopathies, e.g. DBA, is the compound of structure:

Thus, inhibitors of RSK signaling, e.g. inhibitors of RSK p90s6K andp70s6K, inhibitors of rsp6, as well as the specific phenothiazinederivatives and (CaM) inhibitor compounds disclosed herein (e.g. BABTA)can be used in a method for treatment of subjects with ribosomal proteindisorders or ribosomopathies, e.g. Diamond Blackfan anemia (DBA) andother ribosomopathies, such as myelodysplasia, including5q-myelodysplasia, Shwachman-Diamond syndrome and Treacher CollinsSyndrome in human subjects.

Accordingly, one aspect of the present invention relates to a method oftreating a subject with a ribosomal disorder or ribosomopathy,comprising administering an effective amount of an inhibitor ofRSK(p90S6k) to the subject to decrease RSK(p90s6K) activity and decreaseactive p53 in at least one of CD34+ cells, erythroid cells or erythroiddifferentiated cells in the subject. In one embodiment, the inhibitor ofp90S6k inhibits a variant selected from the group consisting of RSK1,RSK2 and RSK3. In one embodiment, the inhibitor of p90s6K selectivelyinhibits RSK2. Any inhibitor p90s6K is useful in methods of theinvention, e.g. the inhibitor may be a nucleic acid (e.g. DNA or RNA,such as RNAi or shRNA, etc.), or a small molecule compound, or aprotein, e.g. a peptide or antibody, or fragment of an antibody. In oneembodiment, the inhibitor of p90S6k is a compound selected from thegroup consisting of: SL0101 (SL) or a derivative or analogue of SL;BI-D1870 (BI), or a derivative or analogue of BI; or SK, or a derivativeor analogue of the compound.

In another aspect of the invention, a method of treating a subject witha ribosomal disorder or ribosomopathy is provided, the method comprisesadministering an effective amount of an inhibitor of p70S6K to thesubject to decrease p70S6K activity and decrease active p53 in at leastone of CD34+ cells, erythroid cells or erythroid differentiated cells inthe subject. Any inhibitor p70S6K is useful in methods of the invention,e.g. the inhibitor can be a nucleic acid (e.g. DNA or RNA such as RNAi,or shRNA, etc.), or can be small molecule compound, or a protein, e.g. apeptide or antibody, or fragment thereof. In one embodiment theinhibitor of p70S6k is the compound PF-4708671 (PF), or a derivative oranalogue of the compound.

In another aspect, a method of treating a subject with a ribosomaldisorder or ribosomopathy is provided, the method comprisesadministering an effective amount of an inhibitor of Chk2 to the subjectto decrease active p53 in at least one of CD34+ cells, erythroid cellsor erythroid differentiated cells in the subject, wherein the inhibitorof Chk2 comprises a compound selected from the group consisting of CCTand III, or derivatives thereof.

In still another aspect, a method of treating a subject with a ribosomaldisorder or ribosomopathy is provided that comprises administering aneffective amount of an inhibitor of calmodulin to the subject todecrease active p53 in at least one of CD34+ cells, erythroid cells orerythroid differentiated cells in the subject, wherein the inhibitor ofcalmodulin is a phenothiazine compound, or a derivative or analogue ofthe phenothiazine compound, wherein the phenothiazine compound isselected from the group consisting of ACV-1-235 (ACV); JJM-II-221E(221E); and DB1026(PerSucc) DB-4-088-2 (088-2); DB-4-088-3 (088-3);DB-4-086 (086); DB-4-087-2 (087-2); DB-4-087-3 (087-3); DB-4-089 (089),or a compound of Formual I or Formula II.

Another aspect provided, is a method of treating a subject with aribosomal disorder or ribosomopathy, comprising administering aneffective amount of a calcium channel blocker BAPTA-AM or derivative oranalogue thereof to the subject to decrease active p53 in at least oneof CD34+ cells.

In some embodiments of all aspects of the present invention, the methodcomprises treating a subject with a ribosomal disorder where the subjecthas Diamond Blackfan Anemia (DBA) or inherited erythroblastopenia, forexample, where the subject has DBA1, DBA2, DBA3, DBA4, DBA5, DBA6, DBA7,or DBA8. In some embodiments, a subject with a ribosomal disorder has amutation in ribosomal protein 19 (RPS19). In alternative embodiments, asubject with a ribosomal disorder has a mutation in ribosomal proteinfrom at least one of, but not limited to RPS7, RPS10, RPS19, RPS24,PRS26, RPS17, PRS27L RPS29. RPL35A, PRLS and PPL11.

In some embodiments, a subject with a ribosomal disorder has a mutationin a ribosomal protein selected from the group consisting of: rPL2A,rPL2B, rPL3, rpL4A, rPL4B, rPL7A, rPL7B, rPL10, rPL11, rPL16A, rPL17A,rPL17B, rPL18A, rPL18B, Rp119A, rPL19, rPL25, rPL29, rpL31A, rpL31B,rPL36A, rPL40A, rPS1A, rPS6A, rPS6B, rPS14A, rPS15, rPS19, rPS23B,rPS25A, rPS26B, rPS29, rPS29B and rPS31.

In some embodiments of all aspects of the present invention, the methodfurther comprises administering another therapeutic agent to treat theribosomal protein defect, selected from the group consisting of:corticosteroids, blood transfusions and other treatments known topersons of ordinary skill in the art.

In some embodiments of all aspects of the present invention, theinhibitor administered to the subject increases the number of CD71+erythroid cells in the subject and/or increases hemoglobin levels in thesubject.

In some embodiments of all aspects of the present invention, the methodsand inhibitors disclosed herein can be used to treat a subject with aribosomal disorder, such as DBA has a symptom of macrocytic anemiaand/or craniofacial abnormalities.

In some embodiments of all aspects of the present invention, the methodsand inhibitors and inhibitors disclosed herein can be used to treat asubject with a ribosomopathy such as 5q-myelodysplasia, for example,where the subject has a mutation in Rps14 or decrease in Rps14expression. In some embodiments, a subject with 5q-myelodysplasia hasdysplastic bone marrow.

In some embodiments of all aspects of the present invention, the methodsand inhibitors as disclosed herein can be used to treat a subject with aribosomopathy such as Shwachman-Diamond syndrome, for example, where thesubject has a mutation in Sbds. In some embodiments, a subject withShwachman-Diamond syndrome has one or more symptoms selected frompancreatic insufficiency, bone marrow dysfunction, skeletal deformities.

In some embodiments of all aspects of the present invention, the methodsand inhibitors as disclosed herein can be used to treat a subject with aribosomopathy such as Treacher Collins Syndrome, for example, where thesubject has a mutation in TCOF1 (nucleolar). In some embodiments, asubject with Treacher Collins Syndrome has one or more craniofacialdeformities.

In some embodiments the present invention also provides kits comprisingcompositions comprising the inhibitors as disclosed herein for the usein the methods to treat a subject with a ribosomal protein disorder ordisease or ribosomopathy as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the clinical features, treatments and genetics associatedwith Diamond Blackfan anemia (DBA). More than half the patients have amutation in a ribosomal protein gene (all mutations are heterozygous).There is no effective, targeted treatment available for these patients.

FIG. 2 is a chart that shows the additional ribosomopathies, the mostcommon being 5q-subtype of MDS. Any drug that we find that works in ourDBA models would also help patients with other ribosomopathies.

FIG. 3 is a schematic of the molecular consequences of ribosomal proteindeficiency. Disruption of ribosomal biogenesis leads to p53 activationthrough accumulation of free ribosomal proteins binding and sequesteringthe negative regulator of p53, MDM2. This allows for an accumulation ofactive p53 in the nucleus which can then activate downstream targets ofp53 such as p21 and GADD45.

FIGS. 4A to 4B are Zebra fish potos. FIG. 4A shows that zebrafish with amutation in rps29 have decreased expression of cmyb (hematopoieticmarker), flk1 (endothelial vessel marker) and are anemic, shown by lackof benzidine staining (which stains for hemoglobin (Hb)). Theseribosomal protein fish model DBA. (Taylor, A. M., et al. (2012). Exp.Hematol. 40, 228-237.e5., Mirabello, L., et al. (2014). Blood 124,24-32.). FIG. 4B shows zebrafish photos of rescued phenotype. Consistentwith other animal models of DBA, crossing the rps29 mutant with a p53mutant rescues these phenotypes, showing hematopoietic and endothelialdefects are mediated through p53.

FIGS. 5A and 5B show zebrafish photos and structures of novel compoundsthat can rescue the Hb defect in our DBA model. specific Chk2inhibitors, CCT and III, can rescue Hb in fish. FIG. 5A zebra fishphotos of A-3, W-7, CCT and III rescue. FIG. 5B chemical structures ofTFP, A-3, W-7, CCT and III compounds that were identified by thechemical screen to rescue Hb in rps 29−/− embryos.

FIG. 6 shows a brief schematic of the function of Calmodulin. Calmodulinis 1) evolutionarily conserved, 2) abundant in the cell, 3) a Ca²⁺sensor, 4) Interacts with proteins, 5) Activates Kinases/phosphatases,6) CaM inhibitors are FDA approved antipsychotics, 7) Trifluoperazine(TFP).

FIGS. 7A to 7C are schmatics and graphs that show the results of ourinhibitors in a human in vitro model of DBA. FIG. 7A schmatic, cordblood derived CD34⁺ cells were expanded in culture for 4 days and theninfected with a lentivirus that contains a hairpin against RPS19 orLuciferase as a control. After 3 days of selection, cells were moved toerythroid differentiation media and incubated with drugs until day 12.Cells were then harvested for Flow cytometry analysis and RNA. On day12, RPS19 deficient cells have only 40% erythroid precursor cells, whileour control cells have 70%. FIG. 7B, % erythroid precursor cells on day12 after TFP treatment. Treating with TFP increases the percentages ofCD71+ cells. RPS19 deficiency increases p21 mRNA levels. FIG. 7C is agraph of p21 levles after TFP treatment. Treatment with TFP dosedependently decreases p21 mRNA. We also see this with our Chk2inhibitors (data not shown). Accordingly, CAM inhibitors rescue human invitro model of DBA and reduce p53 target genen expression.

FIGS. 8A to 8D are schematics and graphs that show TFP partially rescuesanemia in DBA inducible mouse model, using a dox-inducible RPS19knockdown mouse model. (Jaako, P., et al. (2011). Blood 118, 6087-6096.)FIG. 8A, schematic of procedure. WT mice were irradiated andtransplanted with marrow from a donor mouse that contains adox-inducible hairpin against RPS19. After engraftment, mice were feddox to induce Rps19 deficiency and treated with TFP every other day for2 weeks. After 2 weeks, blood and bone marrow are collected foranalysis. FIG. 8B, graph of p21 mRNA Levels. Using p21 mRNA levels for ameasure of p53 activity in the marrow of the mice, Rps19 deficient micehave increased p21 mRNA levels. Treating mice with TFP decreases thelevel of p21 mRNA in the mice. FIG. 8C, graph of red blood cell number.FIG. 8D, graph of Hemoglobin levels. Treating RPS19 deficient mice withTFP significantly increases Red blood cell number and Hemoglobin levels.Thus compounds found in a zebrafish chemical screen were able to rescuea human in vitro and mammalian in vivo model of DBA.

FIGS. 9A to 9C shows schematics and graphs. FIG. 9A a schematic of wildtype P53 and deleted regulatory region p53 (p53 reg). FIG. 9B Schmaticof procedure. Saos2 cells (p53-null cells) transiently transfected withp53 constructs and were treated with vehicle or 20 μM TFP for 24 hours.TFP activity on p53 was assessed by the ability of TFP to reduce p21mRNA levels measured by qPCR. FIG. 9C is a graph of p21 RNA expression.Since our compound decreases p53 activity, we wanted to determine theregion of p53 is responsible for CaM inhibitor activity. Mutation of thenuclear localization sequence had no effect on TFP activity and TFP didnot inhibit the ability of p53 to form a tetramer. Next, the c-terminal30 amino acids from p53 (363-393) were removed. This construct isp53-reg. We found that the regulatory region (REG) of p53 is requiredfor TFP activity.

FIGS. 10A to 10C show schematics, graphs and gels that indictate CaM andChk2 inhibitors reduce S392 phosphorylation. FIG. 10A is a schematic ofconsereved regulatory region phosphorylation sites. FIG. 10B are graphsof an unbiased mass spectrometry analysis looking for post-translationalmodifications of p53 altered in the presence of the CaM inhibitor TFP orthe Chk2 inhibitor, BML. 293T cells were transfected with WT-p53 taggedwith GFP and treated with TFP or BML for 3 hours. P53 wasimmunoprecipiated using an antibody against GFP and was submitted formass spec analysis. Surprisingly, only 1 residue was found to bedifferentially phosphorylated in the presence of TFP and BML. By twodifferent mass spec methods, either conventional mass spec or by TMTtagging of the peptide we found in both cases the percentage of peptidescontaining phosphorylated serine 392 was reduced in TFP and BML treatedsamples (FIG. 10B). It was also noted that this serine is conserved inboth zebrafish and mouse (FIG. 10A). FIG. 10C is a Western Blot thatindicates phosphorylation decrease. Serine 6 and Serine 315 wereidentified to be phosphorylated, but the percentage of peptidescontaining these phosphorylations did not change with TFP or BMLtreatment.

FIGS. 11A to 11C are schematics and graphs which indicate that p53 S392phhosphomimetic prevents TFP from reducing p53 activity. To confirm theimportance of the ability of CaM inhibitors to block the phosphorylationof p53, we created phosphomimetic p53 by replacing S392 with an asparticacid, which mimics constitutive phosphorylation. We also generated acontrol phosphomimetic p53 where S15 was replaced with aspartic acid.This mutant should have no effect on the ability of TFP to reduce p53activity. FIG. 11A is a schematic of experiment used to test thesemutants, we transiently transfected, WT p53, S15D and S392D into Saos2cells and treated them with vehicle or TFP. To assess p53 activity, weexamined p21 and MDM2 mRNA levels. FIG. 11B is a graph of p21Expression. FIG. 11C is a graph of MDM2 mRNA expression. In cells thatcontain WT p53 or S15D p53, TFP was able to reduce p21 and MDM2 levels.However, in cells that contained p53 S392D, TFP was unable to reduce p21or MDM2 levels. Confirming that blocking the S392 phosphorylation isimportant for the activity of TFP.

FIG. 12 shows a graph of percent inhibition of the in vitro kinaseactivity of CaM and Chk2 inhibitors. We asked if CaM and Chk2 inhibitorsare blocking specific kinases. The known kinases that phosphorylate S392are CK2 and p38. However the inhibitors for these kinases did not rescueHb in rps29−/− fish. Thus, the question became what is TFP inhibitingupstream? Is it inhibiting a kinase that phosphorylates S392? Wesubmitted CaM and Chk2 inhibitors for Kinase profiling using ThermoFisher's SelectScreen. Through screening 100 kinases, we found thatmultiple CaM and Chk2 inhibitors blocked the activity of multiplekinases in the ribosomal s6 protein kinase family both p70S6K and p90S6K(RSK)s.

FIG. 13 shows a graph of percent inhibition of the in vitro kinaseactivity of CaM and Chk2 inhibitors on members of the RSK family, RSk1(p90S6K), RSK 2 (p90S6K), RSK3 (p90S6K), RSK4, (p90S6K) and p70S6K.

FIG. 14 is a schematic that shows literature evidence for CaM playing arole in translation and RSK pathway. The following literature is cited:RSK inhibitors were found in a screen of 23K shRNAs for p53 inhibitorsthat reduce p53 activity (p21 levels) but not protein levels Berns, K.et al. (2004). Nature 428, 431-437; Increased Calcium or ionomycintreatment causes sustained RSK activation Chuderland, D., Marmor, G.,Shainskaya, A., and Seger, R. (2008). 1 Biol. Chem. 283, 11176-11188; AAacids activate mTOR through Ca/CaM signaling Gulati, P. et al. (2008).Cell Metabolism 7, 456-465; RSK2 and 3 are elevated in rps29^(−/−)microarray compared to WT/HET and p70S6K, downstream of mTOR and RSK,can phosphorylate p53 S392 Ci, Y. et al. (2014). Cell Death and Disease5, e1542-10. We hypothesized that RP deficiency increases RSK signalingand CaM and Chk2 inhibitors are blocking downstream signaling from RSKand asked if RSK activity increased during ribosomal protein deficiency.

FIGS. 15A to 15B are gels and charts that indicate that Rps29−/− embryoshave increased levels or RSK (p90S6k), p70s6K and RPS6 compared torps29+/+ embryos. (phospho-RSK antibody does not work in zebrafish).FIG. 15A gel of embryo lysates. Whole embryos were lysed in RIPA bufferand run on a 10% SDS-PAGE gel. Lane 1. WT=WT/rps29^(+/−) 48hpf embryolysates. Lane 2. MU=rps29^(−/−) 48hpf embryo lysates. FIG. 15B, amicroarray comparing WT and rps29−/− embryos at 24hpf, RSK2 wasidentified as a top differentially expressed gene, with a mean foldchange value of 1.6-fold over WT embryos. Mean fold change values forp53 and MDM2, which are known to be high during rps deficiency, areincluded for comparison.

FIGS. 16A to 16C inhibitors of RSK and p70S6K increase Hb in rps29−/−embryos. FIG. 16A bar graph of Hb levels % embryos vs. SL1010. FIG. 16Bbar graph of Hb levels % embryos vs. SL1010. FIG. 16C representativezebra fish images; SL=RSK inhibitor, PF=p70S6K inhibitor, FLU=CaMinhibitor. The CaM inhibitor, fluphenazine (FLU), rescues better thaneither RSK or p70S6K inhibitor alone.

FIGS. 17A to 17C are scehmatics and gels that indicate CaM and Chk2inhibitors block phosphorylation of p70S6K selectively during RPdeficiency. FIG. 17A schematic, we used 293T cells, and inducedribosomal protein deficiency using shRNA for RPS19 or Luc for control.FIG. 17B a gel indicating that the phosphorylation of p70S6K isselectively inhibited by TFP and BML during ribosomal proteindeficiency. ERK phosphorylation is increased by these inhibitors, whichis also seen with RSK inhibitors. FIG. 17C a gel of the effect of Lucand RPS10 shRNA on phosphorylated S392. CaM and Chk2 inhibitors inhibitthe ribosomal s6 kinase family in human cells. RSK inhibitors increaseERK pjosphorylation through a engative feedback loop. So TFP and BMLsimilarly affect ERK in the same manner.

FIG. 18 is a schematic of the assay of RSK activation in human cells.The most relevant in vitro model in the blood field is primary humancells—such as Peripheral blood mononuclear cells (PBMCs).

FIGS. 19A to 19B shows that RSk is activated upon RPS19 deficiency inCD34 cells. FIG. 19A gel of phosphorylation showing the results from TFPtreatment of RPS19 deficient CD34 cells, indicates that RSK is activatedand RPS6 phosphorylation is increased in RPS19 knockdown cells. FIG. 19Bschematic ov RP deficiency. TFP treatment reduces P-RPS6 and P-p53 S392and p53 total levels.

FIG. 20 is a gel that shows p-70S6K directly phosphorylates p53 at S392within 10 minutes. Using recombinant active p70S6K or RSK and combiningit with recombinant p53 and ATP, we see that only p70S6K canphosphorylate p53 at S392. RSK did not phosphorylate p53 after 30minutes of incubation with ATP. S15, S20 and S315 are not phosphorylatedby p70S6K and RSK1/2/3/ does not phosphorylate p53 in the sameconditions (data not shown).

FIG. 21 is a schematic of our current model: During RP deficiency, thereare not enough functional ribosomes and not enough translation. The cellis trying to upregulate translation (especially in RBCs when they needto make a lot of globin). To compensate, the RSK axis is much moreactive during RP deficiency, in an effort to get more translation.Calcium needs CaM to activate many kinases and for signaling for boththe mTOR axis and the RSK axis. Not to be bound by theory, we proposethat CaM inhibitors are inhibiting these kinases by binding up theavailable CaM and thus blocking the signaling. Chk2 inhibitors areworking directly on inhibiting RSK and p70S6K (shown through our invitro kinase profiling).

FIGS. 22A and 22B show a graph and gel that indicates that indicateintracellular Ca²⁺ levels are increased in RP deficient cells andchelation reduces p70S6K and RS6 phosphorylation. Not all CaM inhibitorsdirectly inhibited RSK or p70S6K in our in vitro kinase profiling. Wehypothesize that the CaM inhibition is indirect, CaM is needed forcalcium signaling. FIG. 22A, a graph of calcium in cell lysates using acolormetric calcium detection kit we measured the amount of calcium incellular lysates and found that cells that had RPS19 knocked down hadincreased levels of calcium and treating those lysates with a calciumchelator, BAPTA-AM, was able to reduce the calcium levels. FIG. 22B is agel of phosphoryaltion, we show that chelating calcium in cells reducesp70S6K and RPS6 phosphorylation, similar to our CaM inhibitors (TF). RSKinhibitor: SL and B1. P70s6K inhibitor: PF. In Summary we show: 1) CaMand Chk2 inhibitors reduce S392 phosphorylation of p53—to a much greaterextent during ribosomal protein deficiency 2) CaM and Chk2 inhibitorsalso decrease P-p70S6K to a much greater extent during RP deficiency 3)p70S6K can directly phosphorylate p53 S392 in vitro 4) Total proteinlevels of RSK, p70S6K and rps6 are increased ruing RP deficiency invitro and in vivo 5) RSK2 is increased in our microarray in rps20−/− vsWT 24hpf embryos. Protein validates this upregulation 6) Calcium levelsare increased during RP deficiency 7) Blocking the CaM-Calcium signaling(CaM inhibitors) or directly inhibiting RSK or p70S6K (Chk2 inhibitors)leads to a decrease in P-p53 and p53 activity. Reduced p53 activityleads to fewer RBCs undergoing apoptosis, alleviating the anemia.

FIG. 23 is a bar graph that indicates FDA-approved phenothiazines suchas perphenizine and fluphenazine increase Hb in the anemic rps29−/−zebrafish embryos. However, long-term exposure to phenothiazines areassociated with negative neurological side effects. Thus we wanted toscreen for phenothiazine derivatives. Our Rational: Phenothiazinesimpair locomotor activity in zebrafish (Boehmler W, et al. Genes, Brainand Behavior. (2007)), we need a compound that will not enter the brain,others are generating phenothiazine derivatives and there is ahigh-throughput screen for BBB exclusion with behavior

FIG. 24 is a schematic of the generation of phenothiazine derivatives 2general synthesis methods to generate the compounds to test were used.Synthesis 1: compounds which retain the active chemotype (thephenothiazine ring system) and are systematically diversified at apermissive site distal to the aliphatic piperazine. In brief we use anefficient and highly parallel biasing library strategy. Synthesis 2:focused library to explore determinants around the tricyclic ring systemfor biological activity

FIGS. 25A to 25D are bar graphs and zebrafish photos that indicate twosample derivatives synthesized increase Hb in rps29−/− embryos:Perpenazine succ (PerSucc) see FIG. 25A and ACV see FIG. 25C. FIG. 25BPersucc treated embryo as compared to DMSO embryo. FIG. 25D ACV treatedembryo as compared to DMSO embryo.

FIG. 26 is a sample photo of a plate of behavior tracking. 6 dpfWTzebrafish were used, 1 fish/well of 96 well plate. Drug was deliveredinto fish water, we added drug to well and tracked movement for 2h. Wethen used a Matlab algorithm to quantitate movement Drugs: PerphenazineSucc (PerSucc): 221E, and ACV. Yellow square highlights anrepresentative fish swimming very erratically.

FIGS. 27A to 27C are heat maps of fish activity over 2 h of recording.221E (FIG. 21B) and Persucc (FIG. 21C) are drugs. DMSO is the controlvehicle (FIG. 21A).

FIGS. 28A to 28B are graph data summarizing the movement of the fishover 2 h with addition of PerSucc at Rest (FIG. 28A) or Activity (FIG.28B). PerSucc treated fish are less active and rest more than DMSOtreated fish.

FIGS. 29A to 29B are graph data summarizing the movement of the fishover 2 h with addition of ACV at Rest (FIG. 29A) or Activity (FIG. 29B).ACV treated fish are more active and rest less than DMSO treated fish

FIGS. 30A to 30B are graph data summarizing the movement of the fishover 2 h with addition of 221E at Rest (FIG. 30A) or Activity (FIG.30B). 221E treated fish have more erratic activity because they restless and move more than DMSO treated fish. They pause and then movereally fast, then pause and do it again. Thus the behavior studiesrepresent a robust and rapid assay that can differentiate zebrafishmovement: more and less activity vs. erratic, and statistics can beobtained with as little as 8 fish per treatment.

FIG. 31 shows the chemical structures of compounds used in someembodiments of the invention.

FIG. 32 shows chemical structures of specific phenothiazine derivatives.

FIG. 33 shows chemical structures of specific phenothiazine derivatives.Note Abbreviated compound name e.g. is DB-4-083=083.

FIGS. 34A to 34B are zebrafish photos of the scoring method forhemoglobin levels. Wildtype leves (FIG. 34A). High, medium and lowlevels are shown in (FIG. 34B).

FIGS. 35A and 35B are bar graphs showing the effect of the variousphenothiazine derivatives on Hb levels in rps −/− embryos. FIG. 35A:083; 084; 086; and 087-2 effects. FIG. 35B: 087-3; 088-2; 088-3; and 089effects.

FIGS. 36A to 36D are graphs of the number of movement with DMSO or drug.FIG. 36A: fluphenazine. FIG. 36B: Ch2 inhbitor. FIG. 36C: 083. FIG. 36D:chemical structure of 083. 083 decreases movement of zebrafish.

FIGS. 37A to 37D are graphs of the number of movement with DMSO or drug.FIG. 36A: fluphenazine. FIG. 37B: Ch2 inhbitor. FIG. 37C: 088-2. FIG.37D: chemical structure of 088-2. 088-2 does not alter movement ofzebrafish.

FIGS. 38A to 38D are graphs of the number of movement with DMSO or drug.FIG. 38A: fluphenazine. FIG. 38B: Ch2 inhbitor. FIG. 38C: 089. FIG. 38D:chemical structure of 089. 089 alters movement of zebrafish.

FIGS. 39A to 39D are graphs of the number of movement with DMSO or drug.FIG. 39A: fluphenazine. FIG. 39B: Ch2 inhbitor. FIG. 39C: 088-3. FIG.39D: chemical structure of 088-3. 088-3 alters movement of zebrafish.

FIG. 40 is a schematic of the testing of novel phenothiazine derivativesin a human in vitro model of DBA. Primary human peripheral bloodmononuclear cells (PBMCs) are transduced with RPS19-GFP shRNA to make aribiosomal protein deficiency. Cells are transferred to eyrthroiddifferentiation media and treated once for 5 days with Drug. Cells areharvested and analyzed for erythroid precursors by flow cytomety forCD71⁺ cells.

FIG. 41 Flow cytometry charts for CD71⁺ cells in the presence of variousphenothiazine derivatives as indicated in FIG. 40.

FIG. 42 is a bar graph depicting the absolute number of erythroid cellscompared to DMSO treated cells. The phenothiazine derivatives increasethe absolute number of erythroid cells.

FIG. 43 A chart that includes a brief summary of the effects of thephenothiazine derivatives on Hb, behavior and Red Blood Cell (RBC)differentiation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, upon the discovery that RSKsignaling is upregulated in ribosomal protein deficient cells, e.g.RPA19 deficient cells. The inventors have discovered that RSK isactivated upon RPS19 deficiency in CD34 cells and that inhibitors ofRSK(p90s6K) as well as inhibitors of p70s6K increase hemoglobin (Hb) inrps29−/− zebrafish embryos, an in vivo model of ribosomal proteindefect. The inventors have further determined that inhibitors of Chk2rescue the Hb in rps29−/− embryos.

Accordingly, the invention relates to the use of novel classes ofcompounds, i.e. inhibitors of RSK (p90S6K), e.g. SL; inhibitors ofp70S6K, e.g. PF; and inhibitors of rps6, to treat ribosomal disordersand ribosomopathies. In some embodiments, the invention relates to theuse of specific Chk2 inhibitors, i.e. CCT and III, for treatment ofribosomal disorders and ribosomapathies, e.g. DBA. In some embodiments,the invention relates to the use of specific phenothiazine derivatives,e.g. PerSucc, ACV, and 221E, DB-4-088-2 (088-2); DB-4-088-3 (088-3);DB-4-086 (086); DB-4-087-2 (087-2); DB-4-087-3 (087-3); DB-4-089 (089),tri-fluoronated substituent derivatives, or a compound of Formual I orFormula II, to treat ribosomal disorders and ribosomopathies, e.g. DBA.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

Definitions

The term “regulate” used herein in reference to expression of a gene,refers to producing an effect on, for example, gene expression. In someembodiments, the effect can be stimulatory, such as increasingexpression of a gene. In some embodiments, the effect can be inhibitory,such as decreasing expression of a gene. The terms “regulate” and“modulate” are interchangeably used herein.

The terms “calmodulin inhibitor” used interchangeably herein, generallyrefers to an agent or molecule that inhibits the activity or expressionof calmodulin. Calmodulin inhibitors can be of synthetic or biologicalorigins. They can be organic, or inorganic molecules, or peptides,antibodies or antisense RNA that inhibit calmodulin. Inhibitors ofcalmodulin of the invention are chemical entities or molecules that caninhibit expression of calmodulin and/or biological activity ofcalmodulin, for example, compounds of TFP and FLU, prodrugs, derivativesand pharmaceutically acceptable salts thereof, have previously beendetermined to be useful in treatment of ribosomal disorders (See USpublication 2015/0265627). Assays for monitoring Calmodulin activity areknown in the art, See for example US publication 2015/0265627. Herein,the inventors have identified, previously undisclosed compounds thatthat work particularly well for treatment of ribosomal disorders, e.g.ACV, 221E and PerSucc, DB-4-088-2 (088-2); DB-4-088-3 (088-3); DB-4-086(086); DB-4-087-2 (087-2); DB-4-087-3 (087-3); DB-4-089 (089),tri-fluoronated substituent derivatives, or a compound of Formual I orFormula II (see FIGS. 25 and 32).

The term “ribosomal protein”, are also referred to herein as“r-proteins” refers to any of the intracellular ribonucleoproteinparticles concerned with protein synthesis; they consist of reversiblydissociable units and are found either bound to cell membranes or freein the cytoplasm. They may occur singly or occur in clusters(polyribosomes). They may occur singly or in clusters, calledpolyribosomes or polysomes, which are ribosomes linked by mRNA and areactively engaged in protein synthesis. Ribonucleoproteins (oftenreferred to as “RNPs”) are important in protein synthesis; they consistof two, one large (L) and one small (S), reversibly dissociable units(called also 60S and 40S subunits in eukaryotes (50S and 30S inbacteria)). The term includes any of the proteins that, in conjunctionwith rRNA, make up the ribosomal subunits involved in the cellularprocess of translation. The term encompasses proteins of the small (S)subunit and the large (L) subunit of the ribosomes. Due to the highconservation of both the RNA and proteins moieties of ribosomes and ofthe ribosome biogenesis machinery from yeast and bacteria, a large partof the knowledge about these organic molecules has come from the studyof E. coli ribosomes, and also applies to humans. In the small (30S)subunit of E. coli ribosomes, the proteins denoted S4, S7, S8, S15, S17,S20 bind independently to 16S rRNA. After assembly of these primarybinding proteins, S5, S6, S9, S12, S13, S16, S18, and S19 bind to thegrowing ribosome. These proteins also potentiate the addition of S2, S3,S10, S11, S14, and S21. Protein binding to helical junctions isimportant for initiating the correct tertiary fold of RNA and toorganize the overall structure. Nearly all the proteins contain one ormore globular domains. Moreover, nearly all contain long extensions thatcan contact the RNA in far-reaching regions. Additional stabilizationresults from the proteins' basic residues, as these neutralize thecharge repulsion of the RNA backbone. Protein-protein interactions alsoexist to hold structure together by electrostatic and hydrogen bondinginteractions. Theoretical investigations pointed to correlated effectsof protein-binding onto binding affinities during the assembly process[2]

The term “ribosomal disorder” or “ribosomal protein disorder” refers toa disease or disorder linked to a mutated and/or abnormal function of aribosome protein. It can include a disease due to mutation in aribosomal protein, or a disease due to a decreased level, or partialloss of function, of a ribosomal protein, or alternatively, a diseasedue to an increased level of a ribosomal protein, as compared to anormal healthy control subject. The term ribosomal disorder includesgenetic diseases of ribosomal proteins, including but not limited to,Diamond Blackfan anemia (DBA), myelodysplasia, Shwachman-DiamondSyndrome (SDS) and Treachers Collins Syndrome (TCS).

The term “ribosomopathy” or “ribosomopathies” refers to any disease ormalfunction of ribosomes. Ribosomes are small organelles found in allcells which are involved in the production of proteins by translatingmessenger RNA. A disease or malfunction of ribosomes include (i) diseaseof ribosomal biogenesis proteins, (ii) disease of small nucleolarribonuceloproteins, and (iii) diseases of ribosomal proteins (asdiscussed above in the definition of “ribosomal protein disorder”), andare all reviewed in Freed et al., Mol. Biosyst. 2010; 6(3); 481-493entitled “When ribosomes go bad: diseases of ribosome biogenesis”, whichis incorporated herein in its entirety by reference. Diseases ofribosomal biogenesis proteins include, but are not limited to TreachersCollins syndrome (TCS), male infertility due to a mutation inUTP14c,native American indian childhood cirrhosis (NAIC), Bowen-Conradisyndrome (BCS), alopecia neurological defect and endrocrinopathysyndrome (ANE syndrome), shwachman-diamond syndrome (SDS), candidategene for primary open angle glaucoma (POAG), and modifier ofneurofibromatosis type I (NF1). Diseases of small nucleolarribonuceloproteins include, but are not limited to, Anauxetic dysplasia(AD), cartilage-hair dysplasia (also called metaphyseal chondrodysplaia,McKusick type; CCH), metaphyseal dysplasia without hypotrichosis (MDWH),Dyskeratosis congenita (also called Zinzzer-Engman-Cole syndrome),Hoyeraal-Hreidarsson syndrome (where some cases are severe variants ofDyskeratosis congenita), and Prader-Willi syndrome (PWS)

The term “derivative” as used herein refers to a chemical substancerelated structurally to another, i.e., an “original” substance, whichcan be referred to as a “parent” compound. A “derivative” can be madefrom the structurally-related parent compound in one or more steps. Thegeneral physical and chemical properties of a derivative are alsosimilar to the parent compound.

The term “functional derivative” and “mimetic” are used interchangeablyherein, and refers to compounds which possess a biological activity (inparticular functional biological activity) that is substantially similarto the biological activity of the entity or molecule for which it's afunctional derivative of The term functional derivative is intended toinclude the fragments, variants, analogues or chemical derivatives of amolecule. In certain embodiments, functional derivatives and functionalanalogues of calmodulin inhibitors (e.g., functional analogues of TFP,A-3, W-7, A-7, W-5 and CGS-9343) can be assessed for their biologicalactivity using the assay as disclosed herein, where derivatives andanalogues which inhibit calmodulin would be considered as functionalderivatives or functional analogues of such calmodulin inhibitors.

The term “analog” as used herein refers to an agent that retains thesame, or a substantially similar biological function (i.e., inhibitionof calmodulin) and/or structure as the molecule or chemical orpolypeptide it is an analogue of. Examples of analogs includepeptidomimetics (a peptide analog), peptide nucleic acids (a nucleicacid analog), small and large organic or inorganic compounds, as well asderivatives and variants of a polypeptide or nucleic acid herein.

The term “substantially similar”, when used to define the biologicalactivity of a derivative or analogue of an inhibitor (e.g. inhibitor ofp90S6k, or p70S6K, or Chk2, or calmodulin) as compared to the biologicalactivity of the inhibitor to which it is a derivative or analogue of,means that a particular derivative or analogue differs from the initialparent inhibitor in chemical structure, by one or more groups orelements, including substitutions, deletions, or additions of groups ofelements, the net effect of which is to retain at least some of thebiological activity found in the initial parent inhibitor with respectto inhibition of kinase or other RSK, Chk2 activity and/or expression.Such biological activity of inhibition by a functional derivative oranalogue of can be assessed by one of ordinary skill in the art usingassays well known in the art, for example, in in vitro kinase assays,which measures phosphorylation of substrates.

The term “tissue” is intended to include intact cells, blood, bloodpreparations such as plasma and serum, bones, joints, muscles, smoothmuscles, and organs.

The term “subject” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment. The term “subject”and “individual” are used interchangeably herein, and refer to ananimal, for example a human, to whom treatment, including prophylactictreatment, with the cells according to the present invention, isprovided. The “non-human animals” of the invention include mammals suchas rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-humanprimates.

The terms “a reference sample” or “a reference level” as usedinterchangeably herein refer to a negative control of the condition. Forexample, in the context of treatment, a reference level is the level ifa subject is not treated. In some embodiments, a reference level in thecontext of diagnosis is the level present in a normal healthy subject.The term “normal healthy subject” refers to a subject who has nosymptoms of any diseases or disorders, or who is not identified with anydiseases or disorders, or who is not on any medication treatment, or asubject who is identified as healthy by physicians based on medicalexaminations. In some embodiments, a reference level or sample usedherein refers to the level measured at a previous time point from asubject being treated.

The terms “treat”, “treatment” and “treating” used interchangeably, withrespect to treatment of a disease or disorder, mean preventing thedevelopment of the disease, or altering the course of the disease (forexample, but not limited to, slowing the progression of the disease), orreversing a symptom of the disease or reducing one or more symptomsand/or one or more biochemical markers in a subject, preventing one ormore symptoms from worsening or progressing, promoting recovery orimproving prognosis in a subject who is at risk of the disease, as wellas slowing or reducing progression of existing disease. The termtreating encompasses reducing or alleviating at least one adverse effector symptom of a condition, disease or disorder associated withinappropriate ribosomal protein function. As used herein with respect toa ribosomal protein disorder, the term treating is used to refer to thereduction of a symptom and/or a biochemical marker of a ribosomalprotein disorder by at least 10%., for example a reduction of p21 and/orp53 levels in CD34+ cells in the subject, or a return of hemoglobin backto normal levels, or a restoration or prevention of craniofacialdeformities. For example but are not limited to, a reduction of p21and/or p53 levels in CD34+ cells in the subject, as an illustrativeexample only, by 10%, would be considered effective treatments by themethods as disclosed herein.

As used herein, the term “treating” includes preventing the progressionand/or reducing or reversing at least one adverse effect or symptom of acondition, disease or disorder associated with a ribosomal proteindisorder or ribosomopathy, for example, DBA. Accordingly, in someembodiments, treatment can be prophylactic in terms of completely orpartially preventing a disease or sign or symptom of a ribosomal proteindisorder or ribosomopathy. For example, subjects known to have amutation in ribosomal protein or alternatively, low expression levels ofa specific ribosomal protein, can be subjected to prophylactic treatmentto prevent the onset of one or more symptoms associated with such amutation in the ribosomal protein, and/or decreased levels in theribosomal protein. In some embodiments, prophylactic treatment can beadministered to subjects who had prior treatment of a disease associatedwith a ribosomal protein disorder. For example, for subjects who havereceived corticosteroids or blood transfusions for the treatment of DBAand/or other previous treatment to stabilize their DBA can beprophylactically treated (e.g. with a calmodulin inhibitor and/orcalcium channel blocker as disclosed herein).

As used herein, the terms “prevent,” “preventing” and “prevention” referto the avoidance or delay in manifestation of one or more symptoms ormeasurable markers of a disease or disorder. A delay in themanifestation of a symptom or marker is a delay relative to the time atwhich such symptom or marker manifests in a control or untreated subjectwith a similar likelihood or susceptibility of developing the disease ordisorder. The terms “prevent,” “preventing” and “prevention” include notonly the complete avoidance or prevention of symptoms or markers, butalso a reduced severity or degree of any one of those symptoms ormarkers, relative to those symptoms or markers arising in a control ornon-treated individual with a similar likelihood or susceptibility ofdeveloping the disease or disorder, or relative to symptoms or markerslikely to arise based on historical or statistical measures ofpopulations affected by the disease or disorder. By “reduced severity”is meant at least a 10% reduction in the severity or degree of a symptomor measurable disease marker, relative to a control or reference, e.g.,at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or even100% (i.e., no symptoms or measurable markers).

The term “prophylactic” or “therapeutic” treatment refers toadministration to the host of one or more of the subject compositions.If it is administered prior to clinical manifestation of the unwantedcondition (e.g., disease or other unwanted state of the host animal)then the treatment is prophylactic, i.e., it protects the host againstdeveloping the unwanted condition, whereas if administered aftermanifestation of the unwanted condition, the treatment is therapeutic(i.e., it is intended to diminish, ameliorate or maintain the existingunwanted condition or side effects therefrom).

As used herein, “gene silencing” or “gene silenced” in reference to anactivity of an RNAi molecule, for example a siRNA or miRNA refers to adecrease in the mRNA level in a cell for a target gene (e.g. p90S6kgene, or p70S6K gene by at least about 5%, about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 99%, about 100% of the mRNA level found in the cellwithout the presence of the miRNA or RNA interference molecule. In onepreferred embodiment, the mRNA levels are decreased by at least about70%, about 80%, about 90%, about 95%, about 99%, about 100%.

As used herein, the term “RNAi” refers to any type of interfering RNA,including but not limited to, siRNAi, shRNAi, endogenous microRNA andartificial microRNA. For instance, it includes sequences previouslyidentified as siRNA, regardless of the mechanism of down-streamprocessing of the RNA (i.e. although siRNAs are believed to have aspecific method of in vivo processing resulting in the cleavage of mRNA,such sequences can be incorporated into the vectors in the context ofthe flanking sequences described herein). The term “RNAi” can includeboth gene silencing RNAi molecules, and also RNAi effector moleculeswhich activate the expression of a gene. By way of an example only, insome embodiments RNAi agents which serve to inhibit or gene silence areuseful in the methods, kits and compositions disclosed herein to inhibitthe RSK p70S6K and p90S6K genes.

As used herein, a “siRNA” refers to a nucleic acid that forms a doublestranded RNA, which double stranded RNA has the ability to reduce orinhibit expression of a gene or target gene when the siRNA is present orexpressed in the same cell as the target gene. The double stranded RNAsiRNA can be formed by the complementary strands. In one embodiment, asiRNA refers to a nucleic acid that can form a double stranded siRNA.The sequence of the siRNA can correspond to the full-length target gene,or a subsequence thereof. Typically, the siRNA is at least about 15-50nucleotides in length (e.g., each complementary sequence of the doublestranded siRNA is about 15-50 nucleotides in length, and the doublestranded siRNA is about 15-50 base pairs in length, preferably about19-30 base nucleotides, preferably about 20-25 nucleotides in length,e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides inlength).

As used herein “shRNA” or “small hairpin RNA” (also called stem loop) isa type of siRNA. In one embodiment, these shRNAs are composed of ashort, e.g. about 19 to about 25 nucleotide, antisense strand, followedby a nucleotide loop of about 5 to about 9 nucleotides, and theanalogous sense strand. Alternatively, the sense strand can precede thenucleotide loop structure and the antisense strand can follow.

The terms “microRNA” or “miRNA” are used interchangeably herein areendogenous RNAs, some of which are known to regulate the expression ofprotein-coding genes at the posttranscriptional level. EndogenousmicroRNAs are small RNAs naturally present in the genome that arecapable of modulating the productive utilization of mRNA. The termartificial microRNA includes any type of RNA sequence, other thanendogenous microRNA, which is capable of modulating the productiveutilization of mRNA. MicroRNA sequences have been described inpublications such as Lim, et al., Genes & Development, 17, p. 991-1008(2003), Lim et al Science 299, 1540 (2003), Lee and Ambros Science, 294,862 (2001), Lau et al., Science 294, 858-861 (2001), Lagos-Quintana etal, Current Biology, 12, 735-739 (2002), Lagos Quintana et al, Science294, 853-857 (2001), and Lagos-Quintana et al, RNA, 9, 175-179 (2003),which are incorporated by reference. Multiple microRNAs can also beincorporated into a precursor molecule. Furthermore, miRNA-likestem-loops can be expressed in cells as a vehicle to deliver artificialmiRNAs and short interfering RNAs (siRNAs) for the purpose of modulatingthe expression of endogenous genes through the miRNA and or RNAipathways.

As used herein, “double stranded RNA” or “dsRNA” refers to RNA moleculesthat are comprised of two strands. Double-stranded molecules includethose comprised of a single RNA molecule that doubles back on itself toform a two-stranded structure. For example, the stem loop structure ofthe progenitor molecules from which the single-stranded miRNA isderived, called the pre-miRNA (Bartel et al. 2004. Cell 116:281-297),comprises a dsRNA molecule.

The term “gene” used herein can be a genomic gene comprisingtranscriptional and/or translational regulatory sequences and/or acoding region and/or non-translated sequences (e.g., introns, 5′- and3′-untranslated sequences and regulatory sequences). The coding regionof a gene can be a nucleotide sequence coding for an amino acid sequenceor a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA andantisense RNA. A gene can also be an mRNA or cDNA corresponding to thecoding regions (e.g. exons and miRNA) optionally comprising 5′- or 3′untranslated sequences linked thereto. A gene can also be an amplifiednucleic acid molecule produced in vitro comprising all or a part of thecoding region and/or 5′- or 3′-untranslated sequences linked thereto.

The term “gene product(s)” as used herein refers to include RNAtranscribed from a gene, or a polypeptide encoded by a gene ortranslated from RNA.

The terms “lower”, “reduced”, “reduction” or “decrease”, “down-regulate”or “inhibit” are all used herein generally to mean a decrease by astatistically significant amount. However, for avoidance of doubt,“lower”, “reduced”, “reduction” or “decrease” or “inhibit” means adecrease by at least 10% as compared to a reference level, for example adecrease by at least about 20%, or at least about 30%, or at least about40%, or at least about 50%, or at least about 60%, or at least about70%, or at least about 80%, or at least about 90% or up to and includinga 100% decrease (i.e. absent level as compared to a reference sample),or any decrease between 10-100% as compared to a reference level. When“decrease” or “inhibition” is used in the context of the level ofexpression or activity of a gene or a protein, e.g. calmodulin, itrefers to a reduction in protein or nucleic acid level or activity in acell, a cell extract, or a cell supernatant. For example, such adecrease may be due to reduced RNA stability, transcription, ortranslation, increased protein degradation, or RNA interference. In someembodiments, a calmodulin inhibitor which is a small-molecule asdisclosed herein can decrease the activity or expression of calmodulin.Preferably, this decrease is at least about 5%, at least about 10%, atleast about 25%, at least about 50%, at least about 75%, at least about80%, or even at least about 90% of the level of expression or activityunder control conditions. The term “level” as used herein in referenceto calmodulin refers to expression or activity of calmodulin.

The terms “up-regulate”,“increase” or “activate” are all used herein togenerally mean an increase by a statically significant amount; for theavoidance of any doubt, the terms “up-regulate”, “increase” or “higher”means an increase of at least 10% as compared to a reference level, forexample an increase of at least about 20%, or at least about 30%, or atleast about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or a 100%increase or more, or any increase between 10-100% as compared to areference level, or an increase greater than 100%, for example, anincrease at least about a 2-fold, or at least about a 3-fold, or atleast about a 4-fold, or at least about a 5-fold or at least about a10-fold increase, or any increase between 2-fold and 10-fold or greateras compared to a reference level. When “increase” is used in the contextof the expression or activity of a gene or protein, it refers to apositive change in protein or nucleic acid level or activity in a cell,a cell extract, or a cell supernatant. For example, such an increase maybe due to increased RNA stability, transcription, or translation, ordecreased protein degradation. Preferably, this increase is at least 5%,at least about 10%, at least about 25%, at least about 50%, at leastabout 75%, at least about 80%, at least about 100%, at least about 200%,or even about 500% or more over the level of expression or activityunder control conditions.

The terms “significantly different than,”, “statistically significant,”and similar phrases refer to comparisons between data or othermeasurements, wherein the differences between two compared individualsor groups are evidently or reasonably different to the trained observer,or statistically significant (if the phrase includes the term“statistically” or if there is some indication of statistical test, suchas a p-value, or if the data, when analyzed, produce a statisticaldifference by standard statistical tests known in the art).

A “pharmaceutical composition” refers to a chemical or biologicalcomposition suitable for administration to a mammalian subject. Suchcompositions may be specifically formulated for administration via oneor more of a number of routes, including but not limited to, oral,parenteral, intravenous, intraarterial, subcutaneous, intranasal,sublingual, intraspinal, intracerebroventricular, and the like.

The term “effective amount” is used interchangeably with the term“therapeutically effective amount” and refers to the amount of at leastone agent, e.g., calmodulin inhibitor and/or calcium channel blocker ofa pharmaceutical composition, at dosages and for periods of timenecessary to achieve the desired therapeutic result, for example, toreduce or stop at least one symptom of the ribosomal disorder orribosomopathy, for example a symptom of high levels of p53 and/or p21 inCD34+ cells in the subject. For example, an effective amount using themethods as disclosed herein would be considered as the amount sufficientto reduce a symptom of the ribosomal disorder or ribosomopathy by atleast 10%. An effective amount as used herein would also include anamount sufficient to prevent or delay the development of a symptom ofthe disease, alter the course of a symptom disease (for example but notlimited to, slow the progression of a symptom of the disease), orreverse a symptom of the disease. Accordingly, the term “effectiveamount” or “therapeutically effective amount” as used herein refers tothe amount of therapeutic agent (e.g. the inhibitors of RSK p90 andp70S6K and specific Chk2 and calmodulin inhibitors disclosed herein) ofpharmaceutical composition to alleviate at least one symptom of aribosomal disorder or ribosomopathy, e.g. DBA. Stated another way,“therapeutically effective amount” of an inhibitor as disclosed hereinis the amount of a calmodulin inhibitor or calcium channel blocker whichexerts a beneficial effect on, for example, the symptoms of theribosomal disorder or ribosomopathy. The dosage administered, as singleor multiple doses, to an individual will vary depending upon a varietyof factors, including pharmacokinetic properties of the inhibitor, theroute of administration, conditions and characteristics (sex, age, bodyweight, health, size) of subjects, extent of symptoms, concurrenttreatments, frequency of treatment and the effect desired. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the therapeutic agent are outweighed by thetherapeutically beneficial effects. The effective amount in eachindividual case can be determined empirically by a skilled artisanaccording to established methods in the art and without undueexperimentation. In general, the phrases “therapeutically-effective” and“effective for the treatment, prevention, or inhibition”, are intendedto qualify the an inhibitor as disclosed herein which will achieve thegoal of reduction in the severity of at least one symptom of a ribosomalprotein disease or disorder or ribosomopathy.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation, for example the carrierdoes not decrease the impact of the agent on the treatment. In otherwords, a carrier is pharmaceutically inert. The terms “physiologicallytolerable carriers” and “biocompatible delivery vehicles” are usedinterchangeably.

The terms “administered” and “subjected” are used interchangeably in thecontext of treatment of a disease or disorder. Both terms refer to asubject being treated with an effective dose of pharmaceuticalcomposition comprising an inhibitor of the invention by methods ofadministration such as parenteral or systemic administration.

As used herein, the term “alkyl” means a straight or branched, saturatedaliphatic radical having a chain of carbon atoms. C_(x) alkyl andC_(x)-C_(y)alkyl are typically used where X and Y indicate the number ofcarbon atoms in the chain. For example, C₁-C₆alkyl includes alkyls thathave a chain of between 1 and 6 carbons (e.g., methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, neopentyl,hexyl, and the like). Alkyl represented along with another radical(e.g., as in arylalkyl) means a straight or branched, saturated alkyldivalent radical having the number of atoms indicated or when no atomsare indicated means a bond, e.g., (C₆-C₁₀)aryl(C₀-C₃)alkyl includesphenyl, benzyl, phenethyl, 1-phenylethyl 3-phenylpropyl, and the like.Backbone of the alkyl can be optionally inserted with one or moreheteroatoms, such as N, O, or S.

In preferred embodiments, a straight chain or branched chain alkyl has30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straightchains, C3-C30 for branched chains), and more preferably 20 or fewer.Likewise, preferred cycloalkyls have from 3-10 carbon atoms in theirring structure, and more preferably have 5, 6 or 7 carbons in the ringstructure. The term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having one or more substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

Substituents of a substituted alkyl can include halogen, hydroxy, nitro,thiols, amino, azido, imino, amido, phosphoryl (including phosphonateand phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyland sulfonate), and silyl groups, as well as ethers, alkylthios,carbonyls (including ketones, aldehydes, carboxylates, and esters),—CF3, —CN and the like.

As used herein, the term “alkenyl” refers to unsaturated straight-chain,branched-chain or cyclic hydrocarbon radicals having at least onecarbon-carbon double bond. C_(x) alkenyl and C_(x)-C_(y)alkenyl aretypically used where X and Y indicate the number of carbon atoms in thechain. For example, C₂-C₆alkenyl includes alkenyls that have a chain ofbetween 1 and 6 carbons and at least one double bond, e.g., vinyl,allyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl,2-methylallyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, and the like). Alkenylrepresented along with another radical (e.g., as in arylalkenyl) means astraight or branched, alkenyl divalent radical having the number ofatoms indicated. Backbone of the alkenyl can be optionally inserted withone or more heteroatoms, such as N, O, or S.

As used herein, the term “alkynyl” refers to unsaturated hydrocarbonradicals having at least one carbon-carbon triple bond. C_(x) alkynyland C_(x)-C_(y)alkynyl are typically used where X and Y indicate thenumber of carbon atoms in the chain. For example, C₂-C₆alkynyl includesalkynls that have a chain of between 1 and 6 carbons and at least onetriple bond, e.g., ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,isopentynyl, 1,3-hexa-diyn-yl, n-hexynyl, 3-pentynyl, 1-hexen-3-ynyl andthe like. Alkynyl represented along with another radical (e.g., as inarylalkynyl) means a straight or branched, alkynyl divalent radicalhaving the number of atoms indicated. Backbone of the alkynyl can beoptionally inserted with one or more heteroatoms, such as N, O, or S.

As used herein, the term “halogen” or “halo” refers to an atom selectedfrom fluorine, chlorine, bromine and iodine. The term “halogenradioisotope” or “halo isotope” refers to a radionuclide of an atomselected from fluorine, chlorine, bromine and iodine.

A “halogen-substituted moiety” or “halo-substituted moiety”, as anisolated group or part of a larger group, means an aliphatic, alicyclic,or aromatic moiety, as described herein, substituted by one or more“halo” atoms, as such terms are defined in this application.

As used herein, the term “haloalkyl” refers to an alkyl substituted withone or more “halo” atoms. For example, halo-substituted alkyl includeshaloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g.halosubstituted (C₁-C₃)alkyl includes chloromethyl, dichloromethyl,difluoromethyl, trifluoromethyl (—CF₃), 2,2,2-trifluoroethyl,perfluoroethyl, 2,2,2-trifluoro-1, 1-dichloroethyl, and the like).

The term “aryl” refers to an aromatic 5-8 membered monocyclic, 8-12membered fused bicyclic, or 11-14 membered fused tricyclic ring system.C_(c) aryl and C_(x)-C_(y)aryl are typically used where X and Y indicatethe number of carbon atoms in the ring system. In some embodiments, 1,2, 3, or 4 hydrogen atoms of each ring can be substituted by asubstituent.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively. C_(x)heteroaryl and C_(x)-C_(y)heteroaryl are typically used where X and Yindicate the number of carbon atoms in the ring system. In someembodiments, 1, 2, 3, or 4 hydrogen atoms of each ring may besubstituted by a substituent.

Exemplary aryl or heteroaryl groups include, but are not limited to,pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl,pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl,benzyl, phenyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl,indenyl, naphthyl, phenyl, tetrahydronaphthyl, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro [2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl, and thelike.

Exemplary heteroaryls include, but are not limited to, those derivedfrom benzo[b]furan, benzo[b] thiophene, benzimidazole, imidazo[4,5-c]pyridine, quinazoline, thieno [2,3-c]pyridine,thieno[3,2-b]pyridine, thieno [2, 3-b]pyridine, indolizine,imidazo[1,2a]pyridine, quinoline, isoquinoline, phthalazine,quinoxaline, naphthyridine, quinolizine, indole, isoindole, indazole,indoline, benzoxazole, benzopyrazole, benzothiazole,imidazou,s-alpyridine, pyrazolou,s-alpyridine, imidazo[1,2-a]pyrimidine,imidazo [1,2-c]pyrimidine, imidazo [1,5-a]pyrimidine, imidazo[1,5-c]pyrimidine, pyrrolo [2,3-b]pyridine, pyrrolo [2,3 cj pyridine,pyrrolo [3,2-c]pyridine, pyrrolo [3,2-b]pyridine, pyrrolo[2,3-d]pyrimidine, pyrrolo[3,2-d]pyrimidine, pyrrolo [2,3-b]pyrazine,pyrazolou,s-alpyridine, pyrrolo[1,2-b]pyridazine, pyrrolo[1,2-c]pyrimidine, pyrrolo [1,2-a]pyrimidine, pyrrolo [1,2-a]pyrazine,triazo [1,5-a]pyridine, pteridine, purine, carbazole, acridine,phenazine, phenothiazene, phenoxazine,1,2-dihydropyrrolo[3,2,1-hi]indole, indolizine, pyrido[1,2-a]indole,2(1H)-pyridinone, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro [2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl,phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. Someexemplary heteroaryl groups include, but are not limited to, pyridyl,furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl orthienyl, pyridazinyl, pyrazinyl, quinolinyl, indolyl, thiazolyl,naphthyridinyl, 2-amino-4-oxo-3,4-dihydropteridin-6-yl,tetrahydroisoquinolinyl, and the like.

Aryl and heteroaryls can be optionally substituted with one or moresubstituents at one or more positions with, for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF3, —CN, or thelike.

The term “cyclyl” or “cycloalkyl” refers to saturated and partiallyunsaturated cyclic hydrocarbon groups having 3 to 12 carbons, forexample, 3 to 8 carbons, and, for example, 3 to 6 carbons. C_(x)cyclyland C_(x)-C_(y)cylcyl are typically used where X and Y indicate thenumber of carbon atoms in the ring system. The cycloalkyl groupadditionally can be optionally substituted, e.g., with 1, 2, 3, or 4substituents. C₃-C₁₀cyclyl includes cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl, 2,5-cyclohexadienyl, cycloheptyl,cyclooctyl, bicyclo[2.2.2]octyl, adamantan1-yl, decahydronaphthyl,oxocyclohexyl, dioxocyclohexyl, thiocyclohexyl, 2-oxobicyclo[2.2.1]hept-1-yl, and the like.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively). C_(x)heterocyclyl andC_(x)-C_(y)heterocyclyl are typically used where X and Y indicate thenumber of carbon atoms in the ring system. In some embodiments, 1, 2 or3 hydrogen atoms of each ring can be substituted by a substituent.Exemplary heterocyclyl groups include, but are not limited topiperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl,piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl,perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl,1,4-dioxanyland the like.

The terms “bicyclic” and “tricyclic” refers to fused, bridged, or joinedby a single bond polycyclic ring assemblies.

As used herein, the term “fused ring” refers to a ring that is bonded toanother ring to form a compound having a bicyclic structure when thering atoms that are common to both rings are directly bound to eachother. Non-exclusive examples of common fused rings include decalin,naphthalene, anthracene, phenanthrene, indole, furan, benzofuran,quinoline, and the like. Compounds having fused ring systems can besaturated, partially saturated, cyclyl, heterocyclyl, aromatics,heteroaromatics, and the like.

The term “cyano” means the radical —CN.

The term, “heteroatom” refers to an atom that is not a carbon atom.Particular examples of heteroatoms include, but are not limited tonitrogen, oxygen, sulfur and halogens. A “heteroatom moiety” includes amoiety where the atom by which the moiety is attached is not a carbon.Examples of heteroatom moieties include —N═, —NR^(N)—, —N⁺(O⁻)═, —O—,—S— or —S(O)₂—, —OS(O)₂—, and —SS—, wherein R^(N) is H or a furthersubstituent.

The term “hydroxy” means the radical —OH.

The term “nitro” means the radical —NO₂.

As used herein, the term, “aromatic” means a moiety wherein theconstituent atoms make up an unsaturated ring system, all atoms in thering system are sp² hybridized and the total number of pi electrons isequal to 4n+2. An aromatic ring canbe such that the ring atoms are onlycarbon atoms (e.g., aryl) or can include carbon and non-carbon atoms(e.g., heteroaryl).

As used herein, the term “substituted” refers to independent replacementof one or more (typically 1, 2, 3, 4, or 5) of the hydrogen atoms on thesubstituted moiety with substituents independently selected from thegroup of substituents listed below in the definition for “substituents”or otherwise specified. In general, a non-hydrogen substituent can beany substituent that can be bound to an atom of the given moiety that isspecified to be substituted. Examples of substituents include, but arenot limited to, acyl, acylamino, acyloxy, aldehyde, alicyclic,aliphatic, alkanesulfonamido, alkanesulfonyl, alkaryl, alkenyl, alkoxy,alkoxycarbonyl, alkyl, alkylamino, alkylcarbanoyl, alkylene, alkylidene,alkylthios, alkynyl, amide, amido, amino, amino, aminoalkyl, aralkyl,aralkylsulfonamido, arenesulfonamido, arenesulfonyl, aromatic, aryl,arylamino, arylcarbanoyl, aryloxy, azido, carbamoyl, carbonyl, carbonyls(including ketones, carboxy, carboxylates, CF₃, cyano (CN), cycloalkyl,cycloalkylene, ester, ether, haloalkyl, halogen, halogen, heteroaryl,heterocyclyl, hydroxy, hydroxy, hydroxyalkyl, imino, iminoketone,ketone, mercapto, nitro, oxaalkyl, oxo, oxoalkyl, phosphoryl (includingphosphonate and phosphinate), silyl groups, sulfonamido, sulfonyl(including sulfate, sulfamoyl and sulfonate), thiols, and ureidomoieties, each of which may optionally also be substituted orunsubstituted. In some cases, two substituents, together with thecarbon(s) to which they are attached to, can form a ring.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy, n-propyloxy, iso-propyloxy, n-butyloxy, iso-butyloxy, andthe like. An “ether” is two hydrocarbons covalently linked by an oxygen.Accordingly, the substituent of an alkyl that renders that alkyl anether is or resembles an alkoxyl, such as can be represented by one of—O-alkyl, —O-alkenyl, and —O-alkynyl. Aroxy can be represented by—O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as definedbelow. The alkoxy and aroxy groups can be substituted as described abovefor alkyl.

The term “alkylaryl”, as used herein, refers to an alkyl groupsubstituted with an aryl group. The term “alkylheteroaryl” refers to analkyl group substituted with an heteroaryl.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl, and-S-alkynyl. Representative alkylthio groups include methylthio,ethylthio, and the like. The term “alkylthio” also encompassescycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.“Arylthio” refers to aryl or heteroaryl groups.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents. Exemplary acylgroups include, but are not limited to, (C₁-C₆)alkanoyl (e.g., formyl,acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.),(C₃-C₆)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl,cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.),heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl,pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl,tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) and heteroaroyl(e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl,furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl,benzo[b]thiophenyl-2-carbonyl, etc.). In addition, the alkyl,cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl groupmay be any one of the groups described in the respective definitions.

The term “alkylamino” means a nitrogen moiety having at least onestraight or branched unsaturated aliphatic, cyclyl, or heterocyclylradicals attached to the nitrogen. The term “alkylamino” includes“alkenylamino,” “alkynylamino,” “cyclylamino,” and “heterocyclylamino.”The term “acylamino” means a nitrogen moiety having at least one arylradical attached to the nitrogen. For example —NHaryl, and —N(aryl)₂.The term “heteroarylamino” means a nitrogen moiety having at least oneheteroaryl radical attached to the nitrogen. For example —NHheteroaryl,and —N(heteroaryl)₂. Optionally, two substituents together with thenitrogen can also form a ring. Unless indicated otherwise, the compoundsdescribed herein containing amino moieties can include protectedderivatives thereof. Suitable protecting groups for amino moietiesinclude acetyl, tertbutoxycarbonyl, benzyloxycarbonyl, and the like.

The term “mono- or di-alkylamino” means —NH(alkyl) or —N(alkyl)(alkyl),respectively, such as —NHCH₃, —N(CH₃)₂, and the like. For example,representative amino groups include —NHCH₃, —N(CH₃)₂, —NH(C₁-C₁₀alkyl),—N(C₁-C₁₀alkyl)₂, and the like.

It is noted in regard to all of the definitions provided herein that thedefinitions should be interpreted as being open ended in the sense thatfurther substituents beyond those specified may be included. Hence, a C₁alkyl indicates that there is one carbon atom but does not indicate whatare the substituents on the carbon atom. Hence, a C₁ alkyl comprisesmethyl (i.e., —CH3) as well as —CR_(a)R_(b)R_(c) where R_(a), R_(b), andR_(c) caneach independently be hydrogen or any other substituent wherethe atom alpha to the carbon is a heteroatom or cyano. Hence, CF₃, CH₂OHand CH₂CN are all C₁ alkyls.

Unless otherwise stated, structures depicted herein are meant to includecompounds which differ only in the presence of one or more isotopicallyenriched atoms. For example, compounds having the present structureexcept for the replacement of a hydrogen atom by a deuterium or tritium,or the replacement of a carbon atom by a ¹³C- or ¹⁴C-enriched carbon arewithin the scope of the invention.

A “pharmaceutically acceptable salt”, as used herein, is intended toencompass any compound described herein that is utilized in the form ofa salt thereof, especially where the salt confers on the compoundimproved pharmacokinetic properties as compared to the free form ofcompound or a different salt form of the compound. The pharmaceuticallyacceptable salt form can also initially confer desirable pharmacokineticproperties on the compound that it did not previously possess, and mayeven positively affect the pharmacodynamics of the compound with respectto its therapeutic activity in the body. An example of a pharmacokineticproperty that can be favorably affected is the manner in which thecompound is transported across cell membranes, which in turn maydirectly and positively affect the absorption, distribution,biotransformation and excretion of the compound. While the route ofadministration of the pharmaceutical composition is important, andvarious anatomical, physiological and pathological factors cancritically affect bioavailability, the solubility of the compound isusually dependent upon the character of the particular salt formthereof, which it utilized. One of skill in the art will appreciate thatan aqueous solution of the compound will provide the most rapidabsorption of the compound into the body of a subject being treated,while lipid solutions and suspensions, as well as solid dosage forms,will result in less rapid absorption of the compound.

Pharmaceutically acceptable salts include those derived from inorganicacids such as sulfuric, sulfamic, phosphoric, nitric, and the like; andthe salts prepared from organic acids such as acetic, propionic,succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic,salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.See, for example, Berge et al., “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19 (1977), the content of which is herein incorporated by referencein its entirety. Exemplary salts also include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,succinate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. Suitable acids which are capable offorming salts with the compounds of the disclosure include inorganicacids such as hydrochloric acid, hydrobromic acid, perchloric acid,nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, and thelike; and organic acids such as 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid,3-phenylpropionic acid, 4-methylbicyclo[2.2.2]oct-2-ene1-carboxylicacid, 4,4′-mefhylenebis(3-hydroxy-2-ene1-carboxylic acid), acetic acid,anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonicacid, cinnamic acid, citric acid, cyclopentanepropionic acid,ethanesulfonic acid, formic acid, fumaric acid, glucoheptonic acid,gluconic acid, glutamic acid, glycolic acid, heptanoic acid,hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid,malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconicacid, naphthalene sulfonic acid, o-(4-hydroxybenzoyl)benzoic acid,oxalic acid, p-chlorobenzenesulfonic acid, propionic acid,p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid,succinic acid, sulfanilic acid, tartaric acid, tertiary butylaceticacid, trifluoroacetic acid, trimethylacetic acid, and the like. Suitablebases capable of forming salts with the compounds of the disclosureinclude inorganic bases such as sodium hydroxide, ammonium hydroxide,sodium carbonate, calcium hydroxide, potassium hydroxide and the like;and organic bases such as mono-, di- and tri-alkyl and aryl amines(e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine,N-methylglucamine, pyridine, picoline, dicyclohexylamine,N,N′-dibezylethylenediamine, and the like), and optionally substitutedethanol-amines (e.g., ethanolamine, diethanolamine, trierhanolamine andthe like).

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection, infusion and other injection orinfusion techniques, without limitation. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration of a pharmaceutical composition comprising at least oneinhibitor as disclosed herein (e.g. the specific CHK2 and CAMinhibitors, or inhibitors of p70S6K, or p90S6K) such that it enters theanimal's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower activity, e.g. inhibition of p70S6K,p90S6k, Chk2 activiry, calmodulin activity. The term refers tostatistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true. The decision is often made using thep-value.

The term “optional” or “optionally” means that the subsequent describedevent, circumstance or substituent may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., at least one) of the grammatical object of the article.By way of example, “an element” means one element or more than oneelement. Thus, in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise. Thus, for example, reference to apharmaceutical composition comprising “an agent” includes reference totwo or more agents.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation. The term“consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment. As usedherein the term “consisting essentially of ” refers to those elementsrequired for a given embodiment. The term permits the presence ofelements that do not materially affect the basic and novel or functionalcharacteristic(s) of that embodiment of the invention.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean ±1%. The present invention is further explained in detail by thefollowing, including the Examples, but the scope of the invention shouldnot be limited thereto.

This invention is further illustrated by the examples which should notbe construed as limiting. The contents of all references citedthroughout this application, as well as the figures and tables areincorporated herein by reference. All patents and other publicationsidentified are expressly incorporated herein by reference for thepurpose of describing and disclosing, for example, the methodologiesdescribed in such publications that might be used in connection with thepresent invention. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

As used herein, “rps6” refers to ribosomal protein S6 (rpS6), acomponent of the 40S ribosomal subunit thought to be involved inregulating translation. While the true function of rpS6 is currentlyunder investigation, studies have shown that it is involved in theregulation of cell size, cell proliferation, and glucose homeostasis.Studies show that the p70 ribosomal protein S6 kinases (S6K1 and S6K2)and p90 ribosomal protein S6 kinases (RSK) both phosphorylate rpS6 andthat S6K1 and S6K2 predominate this function. Genebank accession,NM_001010 (See e.g., Magnuson B1, et al. (2012). “Regulation andfunction of ribosomal protein S6 kinase (S6K) within mTOR signalingnetworks”. Biochemical Journal 441 (1): 1-21).

P90S6K and P70S6K

Ribosomal s6 kinase (rsk) is a family of protein kinases. There are twosubfamilies of rsk, 1) p90rsk referred to as p90S6K herein, also knownas MAPK-activated protein kinase-1 (MAPKAP-K1), and 2) p70rsk referredto as p70S6K herein, also known as S6-H1 Kinase or simply S6 Kinase. Rskis named for ribosomal protein s6, which is part of the translationalmachinery, but several other substrates have been identified, includingother ribosomal proteins.

Cytosolic substrates of p90S6K include protein phosphatase 1; glycogensynthase kinase 3 (GSK3); Ll CAM, a neural cell adhesion molecule; Sonof Sevenless, the Ras exchange factor; and Myt1, an inhibitor of cdc2,as well as others (Morten Frodin and Steen Gammeltoft. 1999. Role andregulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction.Molecular and Cellular Endocrinology 151(1-2): 65-77). In certainembodiments, p90S6K activity is assessed using these substrates andphosphorylation monitored, e.g. in in vitro assays. RSKS areserine/threonine kinases and are activated by the MAPK/ERK pathway.

RSK (p90S6K) phosphorylation of SOS1 (Son of Sevenless) at Serines 1134and 1161 creates a 14-3-3 docking site. This interaction of phospho SOS1and 14-3-3 negatively regulates Ras-MAPK pathway. p90rsk also regulatestranscription factors including cAMP response element-binding protein(CREB); estrogen receptor-α (ERα); IκBα/NF-κB; and c-Fos (Morten Frodinand Steen Gammeltoft Supra). There are several isoforms, variants, ofthe 90 kDa ribosomal S6 kinase (RSK), i.e. RSK1, RSK2, RSK3, and RSK 4(the mutations of these isoforms are well known in the art, See e.g.Lara et al. ‘A review of the p90-RSK family members: Common functionsand isoform specificity’, Cancer Research 73(17) Sep. 1, 2013, OF1-8).p90S6K human protein sequences include e.g. those found in GenebankAccession numbers Q15418.2 (GI: 20178306); NP_066958.2 (GI: 19923570);NP_001305865.1 (GI: 974576789); NP_001305867.1 (GI: 974576791);NP_002944.2 (GI: 20149547); XP_005246023.1 (GI: 530361302). In certainembodiments, the inhibitor of p90S6K directly inhibits p90S6K bydirectly binding to p90S6K and inhibiting its kinase activity.

p70S6 kinase (p70S6K)

Ribosomal protein S6 kinase beta-1 (S6K1), also known as p70S6 kinase(p70S6K), in humans is encoded by the RPS6KB1 gene. It is aserine/threonine kinase that acts downstream of PIP3 andphosphoinositide-dependent kinase-1 in the PI3 kinase pathway (Chung J,Grammer T C, Lemon K P, Kazlauskas A, Blenis J. (1994). “PDGF- andinsulin-dependent pp70S6k activation mediated byphosphatidylinositol-3-OH kinase”. Nature_370 (6484): 71-75; Chung J,Kuo C J, Crabtree G R, Blenis J. (1992). “Rapamycin-FKBP specificallyblocks growth-dependent activation of and signaling by the 70 kd S6protein kinases.” Cell 69 (7): 1227-1236). mTOR is known tophosphorylate p70S6K at threonine 389 has and correlated with autophagyinhibition in various situations. In certain embodiments, inhibitors ofmTor are the inhibitors of p70S6K. In alternative embodiments, theinhibitor of p70S6K directly inhibits p70S6K by directly binding top70S6K and inhibiting its kinase activity.

Substrates of p70S6K include ribosome protein S6 as well as others.Phosphorylation of S6 induces protein synthesis at the ribosome. Incertain embodiments, p70S6K activity is assessed using these substratesand phosphorylation monitored, e.g. in in vitro assays. p70S6K humanprotein sequences include e.g. those found in Genebank Accession numbersNP_001258971.1; NP_001258972.1; NP_001258973.1; NP_001258989.1;NP_003152.1. Reference mRNA sequences include e.g.NM_001272042;NM_001272043; NM_001272044; NM_001272060; NM_003161.

Inhibitors of p90S6K and p70S6K

Provided herein are methods for treating a subject with a ribosomaldisorder or ribosomopathy, that comprise administering an effectiveamount of an inhibitor of ribosomal s6 kinase, RSK (p90S6k), to thesubject to decrease p90S6K activity and decrease active p53 in at leastone of CD34+ cells, erythroid cells or erythroid differentiated cells inthe subject.

Any inhibitor of p90S6K can be used in methods of the invention. Someknown inhibitors of p90S6K include for example, those described inShiliang Li et al. “Identification of Inhibitors against p90 RibosomalS6 Kinase 2 (RSK2) through Structure-Based Virtual Screening with theInhibitor-Constrained Refined Homology Model” J. Chem. Inf. Model.,2011, 51 (11), pp 2939-2947, incorporated herein by reference in itsentirety. Non-limiting examples of inhibitors are disclosed in thefollowing patents and publications: U.S. Pat. No. 7,60,5241,PCT./US2006/000709. Many are commercially available, e.g. Kempferol,Chemical Name:3,5,7-Trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one; and BRD739,Chemical Name:1-[(2-Phenylethl)amino]-3H-naphtho[1,2,3-de]quinoline-2,7-dione; PF4708671, Chemical name;2-[[4-(5-Ethylpyrimidin-4-yl)piperazin1-yl]methyl]-5-(trifluoromethyl)-1H-benzo[d]imidazole)and SL 0101-1Chemical Name:3-[(3,4-Di-O-acetyl-6-deoxy-α-L-mannopyranosyl)oxy]-5,7-dihydro-2-(4-hydroxyphenyl)-4H-lbenzopyran-4-one,which are available from Tocris Bioscience (Avonmouth, Bristol, BS119QD, United Kingdom); and FMK RSK inhibitor (p90 RSK specific) asdescribed in: TL Nguyen et al. “Targeting RSK: an overview of smallmolecule inhibitors” Anticancer Agents Med. Chem. 2008, 8(7), 710-716,and are available from e.g. Axon Medchem BV, Groningen, Netherlands.

In certain embodiments, the inhibitor of p90S6K is BI-D1870, or aderivative or analogue of BI-D1870 (BI). BI has the molecular formula(MF) C₁₈H1₉FN₄O₂ and is a potent and specific inhibitor of each of thep90 ribosomal S6 kinase (RSK) isoforms in vitro and in vivo. Thus itinhibits RSK1, RSK2, RSK3 and RSK4 in vitro (IC50 values 31 nM, 24 nM,18 nM, and 15 nM, respectively), (available from Axon Medchem BV,Groningen, Netherlands). BI-D1870 (BI) has the following structure:

In certain embodiments, the inhibitor of p90S6k is SL0101 (SL), or aderivative or analogue of SL0101 (SL), wherein SL0101 (SL) has thefollowing structure:

SL is a selective inhibitor of ribosomal S6 kinase (RSK) and hasselectivity for RSK 2, (IC50=89 nM for RSK2). SL Does not inhibitupstream kinases such as MEK, Raf and PKC.

Also provided is a method of treating a subject with a ribosomaldisorder or ribosomopathy, comprising administering an effective amountof an inhibitor of RSK (p70S6K). Any inhibitor of p70S6K can be used inmethods of the invention. Some known inhibitors of p70S6K include forexample, those described in Upul Bandaragea et al.“4-(Benzimidazol-2-yl)-1,2,5-oxadiazol-3-ylamine derivatives: Potent andselective p70S6 kinase inhibitors” Bioorganic & Medicinal ChemistryLetters 19(17), 1 Sep. 2009, Pages 5191-5194; and rapamycin and itsderivatives, and others e.g. described in Sandrine Faivre et al.“Current development of mTOR inhibitors as anticancer agents” NatureReviews Drug Discovery 5, 671-688 (August 2006 each incorporated hereinby reference in its entirety. Many are commercially available, e.g. PF4708671 MF C₁₉H₂₁F₃N₆ RSK inhibitor (p70 RSK specific) (available fromAxon Medchem BV, Groningen, Netherlands); and DG2, MF C₁₆H₁₇BrN₆O, a RSKinhibitor (p70 ribosomal S6 kinase 1 specific).

In certain embodiments, the inhibitor of p70S6k is PF-4708671 (PF), or aderivative or analogue of PF-4708671 (PF), wherein PF-4708671 (PF) hasthe following structure:

The ability of a compound to inhibit the target proteins identifiedherein, e.g. p70S6K, p90SKs, can be assessed by measuring a decrease inkinase activity of the inhibitors as compared to the activity of theproteins in the absence of the respective inhibitor.

Other Inhibitors

Also provided is a method of treating a subject with a ribosomaldisorder or ribosomopathy, comprising administering an effective amountof an inhibitor of Chk2 to the subject to decrease active p53 in atleast one of CD34+ cells, erythroid cells or erythroid differentiatedcells in the subject, wherein the inhibitor of Chk2 comprises a compoundselected from the group consisting of CCT and III, or derivativesthereof, wherein CCT and III have the following structures:

Checkpoint kinases (Chks) (e.g. Chk1 and Chk2) are serine/threoninekinases that are involved in the control of the cell cycle. They areessential components to delay cell cycle progression in normal anddamaged cells and can act at all three cell cycle checkpoints.

As used herein “CCT” refers to CCT 241533 dihydrochloride, availablefrom Tocris biosciences Supra. CCT is a potent Chk2 inhibitor (IC50=3nM). Shows >63-fold selectivity for Chkl over Chk2 and a panel of 84other kinases. CCT inhibits Chk2 activation in response toetoposide-induced DNA damage in HT29 cells and blocks ionizingradiation-induced apoptosis of mouse thymocytes.

Phenothiazine Compounds

Also provided is a method of treating a subject with a ribosomaldisorder or ribosomopathy, comprising administering an effective amountof an inhibitor of calmodulin to the subject to decrease active p53 inat least one of CD34+ cells, erythroid cells or erythroid differentiatedcells in the subject, wherein the inhibitor of calmodulin is aphenothiazine compound, or a derivative or analogue of the phenothiazinecompound, wherein the phenothiazine compound is selected from the groupconsisting of ACV-1-235 (ACV); JJM-II-221E (221E); and DB1026(PerSucc)having the following structures:

Also provided is a method of treating a subject with a ribosomaldisorder or ribosomopathy, comprising administering an effective amountof an inhibitor of calmodulin to the subject to decrease active p53 inat least one of CD34+ cells, erythroid cells or erythroid differentiatedcells in the subject, wherein the inhibitor of calmodulin is aphenothiazine compound, or a derivative or analogue of the phenothiazinecompound, and wherein the phenothiazine compound is selected from thegroup consisting of DB-4-083 (083); DB-4-084 (084); DB-4-088-2 (088-2);DB-4-088-3 (088-3); DB-4-086 (086); DB-4-087-2 (087-2); DB-4-087-3(087-3); DB-4-089 (089) having the following structures:

In certain embodiments, the phenothiazine compound to treat ribosomaldisorders and ribosomopathies, e.g. DBA, is a compound of Formula (I):

wherein:

-   -   X is O or S;    -   R¹ is H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyls, acyl,        aryl, heteroaryl, alkylheteroaryl or alkylaryl;    -   each R is independently H, halo, alkyl, alkyl, alkenyl, alkynyl,        haloalkyl, CN, OH, NH₂, alkylamino, dialkylamino, CO₂H, acyl,        SH, thioalkoxy, SO₂H, or SO₃H; and    -   isomers and pharmaceutically acceptable salts thereof.

In certain embodiments, X is S in compounds of Formula (I). In someother embodiments, X is O in compounds of Formula (I).

The R¹ group in compounds of Formula (I) can be selected from the groupconsisting of H, alkyl, cyclyl, heterocyclyls, acyl, aryl, heteroaryl,alkylheteroaryl and alkylaryl. Optionally, alkyl, cyclyl, heterocyclyls,acyl, aryl, heteroaryl, alkylheteroaryl and alkylaryl of the R¹ groupcan be substituted with 1, 2, 3 or 4 substituents.

In some embodiments, R¹ is a C₁-C₆ alkyl. Exemplary alkyls for the R¹group include, but are not limited to methyl, ethyl, propyl, isopropyl,n-butyl, t-butyl, sec-butyl, iso-butyl, pentyl and hexyl. In someembodiments, R¹ is isopropyl.

In still some other embodiments, R′ is an optionally substitutedalkylaryl, for example a C₁-C₆alkylaryl, which can be optionallysubstituted. Exemplary aryl for the alkylaryl of the R¹ group include,C₁-C₆alkylphenyl. In some embodiments, R¹ is benzyl.

In yet other embodiments, R¹ is a heterocyclyls, optionally substitutedwith one, two, three or four substituents. Exemplary heterocyclyls forthe R¹ group include, but are not limited to, optionally substitutedpiperidinyl. In some embodiments, R¹ is 4-piperidinyl, optionallysubstituted at the N atom. For example, R¹ is N-methylpiperidin-4-yl.

In compounds of Formula (I), the R groups can be same or different andare independently selected from the group consisting of H, halo, alkyl,haloalkyl, CN, OH, NH₂, alkylamino, dialkylamino, CO₂H, acyl, SH,thioalkoxy, SO₂H, and SO₃H. In some embodiments, each R independently isH, halo, C₁-C₆alkyl, C₁-C₆haloalkyl, OH, C₁-C₆alkoxy, NH₂, NO₂,C₁-C₆alkylamino, di(C₁-C₆alkyl)amino, CN or CO₂H.

In some further embodiments, each R independently is H, Cl, F, Br,methyl, trifluoromethyl, OH, NH₂, NO₂, CN or CO₂H.

In some embodiments of compounds of Formula (I), R¹ is alkyl,heterocyclyl, or alkylaryl, each of which can be optionally substituted;and each R is H, halo, alkyl or haloalkyl.

In some embodiments, the phenothiazine compound to treat ribosomaldisorders and ribosomopathies, e.g. DBA, is a compound of Formula II:

wherein:

-   -   each R²¹ is independently selected from the group consisting of        H, halo, alkyl, haloalkyl, CN, OH, NH₂, alkylamino,        dialkylamino, CO₂H, acyl, SH, thioalkoxy, SO₂H, and SO₃H;    -   isomers and pharmaceutically acceptable salts thereof.

In compounds of Formula (II), all R²¹ can be the same, all different ortwo same and one different. In some embodiments, each R²¹ independentlyis H, halo, C₁-C₆alkyl, C₁-C₆haloalkyl, OH, C₁-C₆alkoxy, NH₂, NO₂,C₁-C₆alkylamino, di(C₁-C₆alkyl)amino, CN or CO₂H. For example, each R²¹independently can be H, Cl, F, Br, methyl, trifluoromethyl, OH, NH₂,NO₂, CN or CO₂H.

In one embodiment, the phenothiazine compound to treat ribosomaldisorders and ribosomopathies, e.g. DBA, is the compound of structure:

Also provided is a method of treating a subject with a ribosomaldisorder or ribosomopathy, comprising administering an effective amountof the is a calcium channel blocker BAPTA-AM or derivative or analoguethereof to the subject to decrease active p53 in at least one of CD34+cells wherein BAPTA-AM has the following structure:

RNAi Inhibitors of p90S6K and p70S6K and rbs6

As discussed herein, the inventors have discovered that inhibition ofthe rsk proteins p90S6K and p70S6K and inhibitors of rps6, can be usedin the methods for the treatment of ribosome protein disorders andribosomopathy as disclosed herein. In some embodiments, the inhibitor innot a small molecule compound, but rather a protein inhibitor, and insome embodiments, the inhibitor is any nucleic acid which inhibits thefunction of p90S6K and p70S6K or the expression of p90S6K and p70S6Kfrom its gene. In some embodiments, an inhibitor of p90S6K and p70S6K isa gene silencing agent.

Human p90S6K is also known by aliases; HU-1, MAPKAPK1A, RSK, RSK1,p90Rsk, 90 kDa ribosomal protein S6 kinase, 1MAP kinase-activatedprotein kinase, 1aMAPK-activated protein kinase 1aMAPKAP kinase, isencoded by nucleic acid sequence, chromosome 1, NC_000001.11(26529758..26575029). One homo sapiens ribosomal protein S6 kinase A2(RPS6KA2) variant, i.e., transcript variant 1, is mRNA GenebankNM_021135.5, which is SEQ ID NO: 1 herein, and has an amino acid of SEQID NO: 2. Inhibition of the p90S6K gene can be by gene silencing RNAimolecules according to methods commonly known by a skilled artisan. Forexample, a gene silencing siRNA oligonucleotide duplexes targetedspecifically to human p90S6K can readily be used to knockdown p90S6Kexpression. p90S6K mRNA can be successfully targeted using siRNAs; andother siRNA molecules may be readily prepared by those of skill in theart based on the known sequence of the target mRNA. Accordingly, inavoidance of any doubt, one of ordinary skill in the art can designnucleic acid inhibitors, such as RNAi (RNA silencing) agents to thenucleic acid sequence of SEQ ID NO: 1 which is as follows:

ORIGIN (SEQ ID NO: 1)   1 aggcgtggcg cgtggccggc gctggtactc gcaggggagg cggagaagga ggcggaggga  61 gcgattgtgg ccccggccgc ggtggccggc gcggcctgcc ctttgtgacc gcagctcgcg 121 ccccacgccc cgcgcccatg gccgccgtgc cgggctccct ggccacgcgt gcccgcccgc 181 ggacctgagc cccgcgcctg ggatgccggg gatgcgcgtc ccccggccct gcggctgctc 241 cgggctgggc gcggggcgat ggacctgagc atgaagaagt tcgccgtgcg caggttcttc 301 tctgtgtacc tgcgcaggaa gtcgcgctcc aagagctcca gcctgagccg gctcgaggaa 361 gaaggcgtcg tgaaggagat agacatcagc catcatgtga aggagggctt tgagaaggca 421 gatccttccc agtttgagct gctgaaggtt ttaggacaag gatcctatgg aaaggtgttc 481 ctggtgagga aggtgaaggg gtccgacgct gggcagctct acgccatgaa ggtccttaag 541 aaagccaccc taaaagttcg ggaccgagtg agatcgaaga tggagagaga catcttggca 601 gaagtgaatc accccttcat tgtgaagctt cattatgcct ttcagacgga aggaaagctc 661 tacctgatcc tggacttcct gcggggaggg gacctcttca cccggctctc caaagaggtc 721 atgttcacgg aggaggatgt caagttctac ctggctgagc tggccttggc tttagaccat 781 ctccacagcc tggggatcat ctacagagat ctgaagcctg agaacatcct cctggatgaa 841 gaggggcaca ttaagatcac agatttcggc ctgagtaagg aggccattga ccacgacaag 901 agagcgtact ccttctgcgg gacgatcgag tacatggcgc ccgaggtggt gaaccggcga 961 ggacacacgc agagtgccga ctggtggtcc ttcggcgtgc tcatgtttga gatgctcacg1021 gggtccctgc cgttccaggg gaaggacagg aaggagacca tggctctcat cctcaaagcc1081 aagctgggga tgccgcagtt cctcagtggg gaggcacaga gtttgctgcg agctctcttc1141 aaacggaacc cctgcaaccg gctgggtgct ggcattgacg gagtggagga aattaagcgc1201 catcccttct ttgtgaccat agactggaac acgctgtacc ggaaggagat caagccaccg1261 ttcaaaccag cagtgggcag gcctgaggac accttccact ttgaccccga gttcacagcg1321 cggacgccca cagactctcc tggcgtcccc ccgagtgcaa acgctcatca cctgtttaga1381 ggattcagct ttgtggcctc aagcctgatc caggagccct cacagcaaga tctgcacaaa1441 gtcccagttc acccaatcgt gcagcagtta cacgggaaca acatccactt caccgatggc1501 tacgagatca aggaggacat cggggtgggc tcctactcag tgtgcaagcg atgtgtgcat1561 aaagccacag acaccgagta tgccgtgaag atcattgata agagcaagag agacccctcg1621 gaagagattg agatcctcct gcggtacggc cagcacccga acatcatcac cctcaaggat1681 gtctatgatg atggcaagtt tgtgtacctg gtaatggagc tgatgcgtgg tggggagctc1741 ctggaccgca tcctccggca gagatacttc tcggagcgcg aagccagtga cgtcctgtgc1801 accatcacca agaccatgga ctacctccat tcccaggggg ttgttcatcg agacctgaag1861 ccgagtaaca tcctgtacag ggatgagtcg gggagcccag aatccatccg agtctgcgac1921 ttcggctttg ccaagcagct gcgcgcgggg aacgggctgc tcatgacacc ctgctacacg1981 gccaatttcg tggccccgga ggtcctgaag cgtcaaggct atgatgcggc gtgtgacatc2041 tggagtttgg ggatcctgtt gtacaccatg ctggcaggat ttaccccttt tgcaaatggg2101 ccagacgata cccctgagga gattctggcg cggatcggca gtgggaagta tgccctttct2161 gggggaaact gggactcgat atctgacgca gctaaagacg tcgtgtccaa gatgctccac2221 gtggaccctc atcagcgcct gacggcgatg caagtgctca aacacccgtg ggtggtcaac2281 agagagtacc tgtccccaaa ccagctcagc cgacaggacg tgcacctggt gaagggcgcg2341 atggccgcca cctactttgc tctaaacaga acacctcagg ccccgcggct ggagcccgtg2401 ctgtcatcca acctggctca gcgcagaggc atgaagagac tcacgtccac gcggctgtag2461 cgggtgggac cctggcccca gcgtcccctg ccagcatcct cgtgggctca cagaccccgg2521 cctcggagcc cgtctggcac ccagagtgac cacaagtcca gcagggaggc ggcgcccgcc2581 ctcgccgtgt ccgtgttttc tttttcagcc ccggagaggg tcctgacctg ggggcttctc2641 caagcctcac tgcgccagcc tccccgcccg ctctcttttc tcccaagcga aaccaaatgc2701 gccccttcac ctcgcgtgcc cgtgcgaggc cgggggcttc tttcagagcc cgcgggtcct2761 ctcatacatg gcttctgttt ctgccgagag atctgttttc caattatgaa gccggtcggt2821 ttggtcagac tcccgacacc cacgtcccag gtacccggtg ggaaagtggc agtgcgaggg2881 cgcagccatt ggtggttgca gggccccaga gggctggggt gacctggcat cccggggctc2941 cccacgggct ggatgacggg gttggcactg tggcgtccag gaggagatgc ctggttctgc3001 ccaaaataat ccaaagagcc gtttcctcct cgcccttcag tttttgcctg aggtgctggg3061 tagcccatcc tttcctctgt cccagattca aatgaggagt aagagcccag acgagaggaa3121 ggcaggctgg atctttgcct tgagagctcc gtgtcaccag gatggaaggg ggtgcctctc3181 ggaggagcct gtgtccacct ccagtctcgg ctttccccgg ggggccaagc gcactgggct3241 gccgtctgtc cccagctccc gtggccacac agctatctgg aggctttgca gggagtcgtg3301 ggttctcgca cctgctcagc cctgtgtcgg cttcctgtgt gctcacctaa agctgtggtt3361 ttgctgtgtt cacttcgatt tttctggtct gtggagaaac tgtgaattgg agaaatggag3421 ctctgtggct tcccacccaa accttctcag tccagctgga ggctggaggg agacacaggc3481 cccacccagc agactgaggg gcagaggcac aggtgggagg gcagcggaga tcagcgtgga3541 caggagcgat gcactttgta gatgctgtgg ctttgtgttg cgttttgtgt ctctgttgca3601 cagatctgtt ttttcacact gatccgtatt cccctgggtg tgcacacagg gcgggtgtgg3661 ggcatttagg ccatgctgtg ctctacttca ttgagtaaaa tcgagtgaga ggttccgggc3721 agcaggatcg acgcccagtc cagccggcag agggaacaca cgggtccttc attgtcctgt3781 aagggtgttg aagatgctcc ctggcggccc ccaagcagac tagatgggag gaggcgccgc3841 tcagcccctc accctgcatc actgaagagc ggcgcctctg cagcaagcag ggcttcagga3901 ggtgcccgct ggccacagcc aggttttccc taagaagatg ttattttgtt gggttttgtt3961 ccccctccat ctcgattctc gtacccaact aaaaaaaaaa aaataaagaa aaaatgtgct4021 gcgttctgaa aaataactcc ttagcttggt ctgattgttt tcagacctta aaatataaac4081 ttgtttcaca agctttaatc catgtggatt ttttttttct tagagaacca caaaacataa4141 aaggagcaag tcggactgaa tacctgtttc catagtgccc acagggtatt cctcacattt4201 tctccataga agatgctttt tcccaaggct agaacgactt ccaccatgat qaatttqctt4261 tttaggtctt aattatttca cttcttttta gaaacttagg aagaagtgga taatcctgag4321 gtcacacaat ctgtcctccc agaaatgaac aaaagtcatc accttttctg cttgctacac4381 aggcaacgat tcccccatca gctgcccgga ccctttggcc tggcttggtg tgcaggcctg4441 tctgtttgct taaagtcagt gggttctggt gcagggagtg agaagtgggg gaagtgaaag4501 ggaaagcatc cgtgagaaag cggccacggt tttccctcct tgtgtgccca tggggcacca4561 gctcatggtc tttttcagtc atcccagttt gtacagactt agcttctgaa ctctaagaat4621 gccaaaggga ccgacgagac tccccatcac agcgagctct gtccttacat gtatttgatg4681 tgcatcagcg gaggagaaca ctggcttggc cctgctccgc tgagtgtctg tgaaatacct4741 ctactttccc tcccatatcc agaacaaaat gatacttgac atccttccac aaaagtcagc4801 ctaaagaagt tatggtatca tatgttaaac taagctttca aaaaccttag tgaaatagca4861 agtgactgct ttcaagcagc agtcgacatg taaatgaagg tgttcttaga attcgcattt4921 tgccagctca gcgcacctcc acaacgaatg aaatgctccg tatgatttgc acaaatgaca4981 tagacctccc caaaagttaa ctggctctcc ttcctcacac agttcatcat aacccaaccc5041 cccacccccg ggtcatgaaa atcacagaac ttataaacac attgaaccct agatctcagg5101 cttcctgacc taccgccagt ggccccttgc tggccaccct atagggtcct ccttccctgg5161 cagcccccca tgtgggagaa atacctgatt ctcccaatct gcagtgggag agctttgctg5221 aattccatcc caaagtcaaa catgggcaag aggtgaggat ttcactttta ccctcaagtc5281 cgatttgtct gtgattttaa actaactgtg tatgtattga tgtttggaag attgtttgaa5341 ttttaaagtg ataatagtac ttaatgttat ccagtattgt tcattaaatg gtgttatcct5401 aaagctgcac ttgggatttt tacctaacgc tttactgatt ctctcaagca catggcaaag5461 tttgatttgc actccgttca tttctgacac gttttgctgc ctcctacctt tctaagcgtc5521 atgcaaattc gagaatggag aaggacgctg ccggtccctg agcggtgtgg agagggcgga5581 aggtggactc cagcgcagct tgaggggctg aggacggagg ctgcagcatc tgtgtcgttc5641 tactgagcac gcttctctgc ctcgctcctg actcagcact ttgttcactg gctcagcagt5701 tatgtttaca catcattttt atgttcctgc tttgtaattc atgtttgaga tgggtggcca5761 ctgtacagat atttattacg ctttccagac tttctgaata gatttttttg aataaacatg5821 gttttatgaa gtgtaatctt tttctagcct aacaataaaa aaaaaaaaaa aSEQ ID NO: 1 encodes the following amino acid sequences, i.e. SEQ ID NO: 2:(SEQ ID NO: 2)MDLSMKKFAVRRFFSVYLRRKSRSKSSSLSRLEEEGVVKEIDISHHVKEGFEKADPSQFELLKVLGQGSYGKVFLVRKVKGSDAGQLYAMKVLKKATLKVRDRVRSKMERDILAEVNHPFIVKLHYAFQTEGKLYLILDFLRGGDLFTRLSKEVMFTEEDVKFYLAELALALDHLHSLGIIYRDLKPENILLDEEGHIKITDFGLSKEAIDHDKRAYSFCGTIEYMAPEVVNRRGHTQSADWWSFGVLMFELTGSLPFQGKDRKETMALILKAKLGMPQFLSGEAQSLLRALFKRNPCNRLGAGIDGVEEIKRHPFFVTIDWNTLYRKEIKPPKFPAVGRPEDTFHFDPEFTARTPTDSPGVPPSANAHHLFRGFSFVASSLIQEPSQQDLHKVPVHPIVQQLHGNNIHFTDGYEIKEDIGVGSYSVCKRCVHDATDTEYAVKIIDKSKRDPSEEIEILLRYGQHPNIITLKDVYDDGKFVYLVMELMRGGELLDRILRQRYFSEREADSVLCTITKTMDYLHSQGVVHRDLKPSNILYRDESGSPESIRVCDFGFAKQLRAGNGRIGSGKYALSGGNWDSISDAAKDVVSKMLHVDPHQRLTAMQVLKHPWVVNREYLSPNQLSRQDVHLVKGAMAATYFALNRTPQAPRLEPVLSSNLAQRRGMKRLTSTRL

Human p70S6K is also known by aliases; p70 S6KA; p70(S6K)-alpha;p70-alpha; p70-S6K; PS6K; S6K; S6K-beta-1; S6K1; STK14A; and RPS6KB1 andis encoded by e.g. Homo sapiens ribosomal protein S6 kinase B1(RPS6KB1), transcript variant 1, mRNA Accession: NM_001272060.1 GI:440546415 nucleic acid sequence (SEQ ID NO: 3), and has an amino acid of(SEQ ID NO: 4). Inhibition of the p70S6K gene can be by gene silencingRNAi molecules according to methods commonly known by a skilled artisan.For example, a gene silencing siRNA oligonucleotide duplexes targetedspecifically to human p70S6K can readily be used to knockdown p70S6Kexpression. p70S6K mRNA can be successfully targeted using siRNAs; andother siRNA molecules may be readily prepared by those of skill in theart based on the known sequence of the target mRNA. Accordingly, inavoidance of any doubt, one of ordinary skill in the art can designnucleic acid inhibitors, such as RNAi (RNA silencing) agents to thenucleic acid sequence of NM_001272060 which is as follows:

(SEQ ID NO: 3)   1 gtttggcttc acggaaccct gtacgcatgc tcctacgctg aactttagga gccagtctaa  61 ggcctaggcg cagacgcact gagcctaagc agccggtgat ggcggcagcg gctgtggtgg 121 ctgcggcggg tccgggccca tgaggcgacg aaggaggcgg gacggctttt acccagcccc 181 ggacttccga gacagggaag ctgaggacat ggcaggagtg tttgacatag acctggacca 241 gccagaggac gcgggctctg aggatgagct ggaggagggg ggtcagttaa atgaaagcat 301 ggaccatggg ggagttggac catatgaact tggcatggaa cattgtgaga aatttgaaat 361 ctcagaaact agtgtgaaca gagggccaga aaaaatcaga ccagaatgtt ttgagctact 421 tcgggtactt ggtaaagggg gctatggaaa ggtttttcaa gtacgaaaag taacaggagc 481 aaatactggg aaaatatttg ccatgaaggt gcttaaaaag gcaatgatag taagaaatgc 541 taaagataca gctcatacaa aagcagaacg gaatattctg gaggaagtaa agcatccctt 601 catcgtggat ttaatttatg cctttcagac tggtggaaaa ctctacctca tccttgagta 661 tctcagtgga ggagaactat ttatgcagtt agaaagagag ggaatattta tggaagacac 721 tgcctgcttt tacttggcag aaatctccat ggctttgggg catttacatc aaaaggggat 781 catctacaga gacctgaagc cggagaatat catgcttaat caccaaggtc atgtgaaact 841 aacagacttt ggactatgca aagaatctat tcatgatgga acagtcacac acacattttg 901 tggaacaata gaatacatgg cccctgaaat cttgatgaga agtggccaca atcgtgctgt 961 ggattggtgg agtttgggag cattaatgta tgacatgctg actggagcac ccccattcac1021 tggggagaat agaaagaaaa caattgacaa aatcctcaaa tgtaaactca atttgcctcc1081 ctacctcaca caagaagcca gagatctgct taaaaagctg ctgaaaagaa atgctgcttc1141 tcgtctggga gctggtcctg gggacgctgg agaagttcaa gctcatccat tctttagaca1201 cattaactgg gaagaacttc tggctcgaaa ggtggagccc ccctttaaac ctctgttgca1261 atctgaagag gatgtaagtc agtttgattc caagtttaca cgtcagacac ctgtcgacag1321 cccagatgac tcaactctca gtgaaagtgc caatcaggtc tttctgggtt ttacatatgt1381 ggctccatct gtacttgaaa gtgtgaaaga aaagttttcc tttgaaccaa aaatccgatc1441 acctcgaaga tttattggca gcccacgaac acctgtcagc ccagtcaaat tttctcctgg1501 ggatttctgg ggaagaggtg cttcggccag cacagcaaat cctcagacac ctgtggaata1561 cccaatggaa acaagtggca tagagcagat ggatgtgaca atgagtgggg aagcatcggc1621 accacttcca atacgacagc cgaactctgg gccatacaaa aaacaagctt ttcccatgat1681 ctccaaacgg ccagagcacc tgcgtatgaa tctatgacag agcaatgctt ttaatgaatt1741 taaggcaaaa aaggtggaga gggagatgtg tgagcatcct gcaaggtgaa acgactcaaa1801 atgacagttt cagagagtca atgtcattac atagaacact tcagacacag gaaaaataaa1861 cgtggatttt aaaaaatcaa tcaatggtgc aaaaaaaaac ttaaagcaaa atagtattgc1921 tgaactctta ggcacatcaa ttaattgatt cctcgcgaca tcttctcaac cttatcaagg1981 attttcatgt tgatgactcg aaactgacag tattaagggt aggatgttgc ttctgaatca2041 ctgttgagtt ctgattgtgt tgaagaaggg ttatcctttc attaggcaaa gtacaaaatt2101 gcctataata cttgcaacta aggacaaatt agcatgcaag cttggtcaaa ctttttccag2161 caaaatggaa gcaaagacaa aagaaactta ccaattgatg ttttacgtgc aaacaacctg2221 aatctttttt ttatataaat atatattttt caaatagatt tttgattcag ctcattatga2281 aaaacatccc aaactttaaa atgcgaaatt attggttggt gtgaagaaag ccagacaact2341 tctgtttctt ctcttggtga aataataaaa tgcaaatgaa tcattgttaa ccacagctgt2401 ggctcgtttg agggattggg gtggacctgg ggtttatttt cagtaaccca gctgcaatac2461 ctgtctgtaa tatgagaaaa aaaaaatgaa tctatttaat catttctact tgcagtactg2521 ctatgtgcta agcttaactg gaagccttgg aatgggcata agttgtatgt cctacatttc2581 atcattgtcc cgggcctgca ttgcactgga aaaaaaaatc gccacctgtt cttacaccag2641 tatttggttc aagacaccaa atgtcttcag cccatggctg aagaacaaca gaagagagtc2701 aggataaaaa atacatactg tggtcggcaa ggtgagggag atagggatat ccaggggaag2761 agggtgttgc tgtggcccac tctctgtcta atctctttac agcaaattgg taagattttc2821 agttttactt ctttctactg tttctgctgt ctaccttcct tatatttttt tcctcaacag2881 ttttaaaaag aaaaaaaggt ctattttttt ttctcctata cttgggctac attttttgat2941 tgtaaaaata tttgatggcc ttttgatgaa tgtcttccac agtaaagaaa acttagtggc3001 ttaatttagg aaacatgtta acaggacact atgtttttga aattgtaaca aaatctacat3061 aaatgattta caggttaaaa gaataaaaat aaaggtaact ttacctttct taaatatttc3121 ctgccttaaa gagagcattt ccatgacttt agctggtgaa agggtttaat atctgcagag3181 ctttataaaa atatatttca gtgcatactg gtataataga tgatcatgca gttgcagttg3241 agttgtatca ccttttttgt ttgtctttta taatgtcttc agtctgagtg tgcaaagtca3301 atttgtaata ttttgcaacc ctaggatttt tttaaataga tgctgcttgc tatgttttca3361 aacctttttg agccatagga tccaagccat aaaattcttt atgcatgttg aattcagtca3421 gaaaagagca aggctttgct ttttgaaatt gcaactcaaa tgagatggga tgaaatccta3481 tgacagtaag caaaaacaga accatgaaaa atgattggac atacaccttt tcaattgtgg3541 caataattga aagaatcgat aaaagttcat ctttggacag aaagccttta aaaaaaaaat3601 cactccctct tccccctcct cccttattgc agcagcctac tgagaacttt gactgttgct3661 ggtaaattag aagctacaat aataattaag ggcagaaatt atacttaaaa agtgcagatc3721 cttgttcttt gacaatttgt gatgtctgaa aaaacagaac ccgaaaagct atggtgatat3781 gtacaggcat tatttcagac tgtaaatggc ttgtgatact cttgatactt gttttcaaat3841 atgtttacta actgtagtgt tgactgcctg accaaattcc agtgaaactt atacaccaaa3901 atattcttcc taggtcctat ttgctagtaa catgagcact gtgattggct ggctataacc3961 accccagtta aaccattttc ataattagta gtgccagcaa tagtggcaaa cactgcaact4021 tttctgcata aaaagcatta attgcacagc taccatccac acaaatacat agtttttctg4081 acttcacatt tattaagtga aatttatttc ccatgctgtg gaaagtttat tgagaacttg4141 tttcataaat ggatatccct actatgactg tgaaaacatg tcaagtgtca cattagtgtc4201 acagacagaa agcacacacc tatgcaatat ggcttatcta tatttatttg taaaaatcca4261 agcatagttt aaaatatgat gtcgatatta ctagtcttga gtttctaaga gggttcttta4321 tgttatacca ggtaagtgta taaaagagat taagtgcttt tttttcatca cttgattatt4381 ttctttaaaa tcagctatta caggatattt ttttatttta tacatgctgt tttttaatta4441 aaatataatc actgaagttt actaatttga ttttataagg tttgtagcat tacagaataa4501 ctaaactggg atttataaac cagctgtgat taacaatgta aagtattaat tattgaactt4561 tgaaccagat ttttaggaaa attatgttct ttttccccct ttatggtctt aactaatttg4621 aatccttcaa gaaggatttt tccatactat tttttaagat agaagataat ttgtgggcag4681 gggtggagga tgcatgtatg atactccata aattcaacat tctttactat aggtaatgaa4741 tgattataaa caagatgcat cttagatagt attaatatac tgagccttgg attatatatt4801 taatatagga cctattttga atattcagtt aatcatatgg ttcctagctt acaagggcta4861 gatctaagat tattcccatg agaaatgttg aatttatgaa gaatagattt taaggctttg4921 aaaatggtta atttctcaaa aacatcaatg tccaaacatc tacctttttt cataggagta4981 gacactagca agctggacaa actatcacaa aagtatttgt cacacataac ctgtggtctg5041 ttgctgatta atacagtact ttttcttgtg tgattcttaa cattatagca caagtattat5101 ctcagtggat tatccggaat aacatctgaa agatgggttc atctatgttt gtgtttgctc5161 tttaaactat tgtttctcct atcccaagtt cgctttgcat ctatcagtaa ataaaattct5221 tcagctgcct tattaggagt gctatgaggg taacacctgt tctgcttttc atcttgtatt5281 tagttgactg tattatttga tttcggattg aatgaatgta aatagaaatt aaatgcaaat5341 ttgaatgaac ataaaaaaaa aaaaaaaaSEQ ID NO: 3 encodes SEQ ID NO: 4, which is as follows: (SEQ ID NO: 04)MAGVFDIDLDQPEDAGSEDELEEGGQLNESMDHGGVGPYELGMEHCEKFEISETSVNRGPEKIRPECFELLRVLGKGGYGKVFQVRKVTGANTGKIFAMKVLKKAMIVRNAKDTAHTKAERNILEEVKHPFIVDLIYAFQTGGKLYLILEYLSGGELFMQLEREGIFMEDTACFYLAEISMALGHLHQKGIIYRDLKPENIMLNHQGHVKLTDFGLCKESIHDGTVTHTFCGTIEYMAPEILMRSGHNRAVDWWSLGALMYDMLTGAPPFTGENRKKTIDKILKCKLNLPPYLTQEARDLLKKLLKRNAASRLGAGPGDAGEVQAHPFFRHINWEELLARKVEPPFKPLLQSEEDVSQFDSKFTRQTPVDSPDDSTLSESANQVFLGFTYVAPSVLESVKEKFSFEPKIRSPRRFIGSPRTPVVSPKFSPGDFWGRGASASTANPQTPVEYPMETSGIEQMDVTMSGEASAPLPIRQPNSGPYKKQAFPMISKRPEHLRMNL.

In certain embodiments, rps6 is inhibited. Inhibition of the rps6 genecan be by gene silencing RNAi molecules according to methods commonlyknown by a skilled artisan. For example, a gene silencing siRNAoligonucleotide duplexes targeted specifically to human rps6 (GenBankNo: NM_001010.2) can readily be used to knockdown rps6 expression. Rps6mRNA can be successfully targeted using siRNAs; and other siRNAmolecules may be readily prepared by those of skill in the art based onthe known sequence of the target mRNA. Accordingly, in avoidance of anydoubt, one of ordinary skill in the art can design nucleic acidinhibitors, such as RNAi (RNA silencing) agents to the nucleic acidsequence of SEQ ID NO: 5 which is as follows:

(SEQ ID NO: 5) 1cctcttttcc gtggcgcctc ggaggcgttc agctgcttca agatgaagct gaacatctcc 61ttcccagcca ctggctgcca gaaactcatt gaagtggacg atgaacgcaa acttcgtact 121ttctatgaga agcgtatggc cacagaagtt gctgctgacg ctctgggtga agaatggaag 181ggttatgtgg tccgaatcag tggtgggaac gacaaacaag gtttccccat gaagcagggt 241gtcttgaccc atggccgtgt ccgcctgcta ctgagtaagg ggcattcctg ttacagacca 301aggagaactg gagaaagaaa gagaaaatca gttcgtggtt gcattgtgga tgcaaatctg 361agcgttctca acttggttat tgtaaaaaaa ggagagaagg atattcctgg actgactgat 421actacagtgc ctcgccgcct gggccccaaa agagctagca gaatccgcaa acttttcaat 481ctctctaaag aagatgatgt ccgccagtat gttgtaagaa agcccttaaa taaagaaggt 541aagaaaccta ggaccaaagc acccaagatt cagcgtcttg ttactccacg tgtcctgcag 601cacaaacggc ggcgtattgc tctgaagaag cagcgtacca agaaaaataa agaagaggct 661gcagaatatg ctaaactttt ggccaagaga atgaaggagg ctaaggagaa gcgccaggaa 721caaattgcga agagacgcag actttcctct ctgcgagctt ctacttctaa gtctgaatcc 781agtcagaaat aagatttttt gagtaacaaa taaataagat cagactctg.

Of course on of skill in the art will understand that other variants ofthese exemplified nucleic acid sequences can be targeted.

One of skill in the art will understand that an inhibitor of p90S6K andp70S6K, or rps6, can be any agent which inhibits the function of p90S6Kand p70S6K, such as antibodies, gene silencing RNAi molecules and thelike. Commercial neutralizing antibodies and fragments of antibodiesagainst p90S6K, p70S6K, and rps6, are encompassed for use in the methodsand compositions as disclosed herein.

A person skilled in the art is able to test whether a certain compoundacts as a p90S6K and p70S6K inhibitor. Test systems for p90S6K andp70S6K activity of certain compounds are well known in the art. Forinstance, such test systems are described in Roux et al.‘Phosphorylation of p90 Ribosomal S6 Kinase (RSK) RegulatesExtracellular Signal-Regulated Kinase Docking and RSK Activity’ Mol CellBiol. 2003 Jul; 23(14): 4796-4804; and Sapkota et al. ‘BI-D1870 is aspecific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms invitro and in vivo’ Biochem J. 2007 Jan 1; 401(Pt 1): 29-38. See also,Masuda-Robens et al. ‘Assays for monitoring p70 S6 kinase and RSKactivation’. Methods Enzymol. 2001;333:45-55. There are also commercialassays available in the art, e.g. p70 S6K activity kit—ADI-EKS-470—EnzoLife Sciences (Farmingdale, N.Y.5); and p70 S6K Activity Kit (ab139438)from Abcam (Cambridge, Mass.).

In addition, as described herein, the ability to rescue at least one ofthe morphological, hematopoietic or endothelial defects in the Rps29 −/−zebrafish embryo and/or prevent p53 function and nuclear accumulation inA549 lung cancer cell line that have had RPS19 knocked down by siRNA, orreduce p21 levels or increase erythroid markers in CD34+ cells that havehad RPS19 knocked down by siRNA is a means for monitoring activity ofthe inhibitors of p90S6K and p70S6K.

In some embodiments, the specific calmodulin inhibitor as disclosedherein (e.g. the phenothiazine derivatives of FIG. 24 or FIG. 33, orPerSucc, or 221E, or ACV of FIG. 32, or the compounds of Formula I andformula II and analogues or derivative thereof can be assessed by one ofordinary skill in the art using assays well known in the art, forexample, inhibition of calmodulin may, inter alia, be determined in thefollowing in vitro assay, which measured the calmodulin-dependentactivation of myosin light chain kinase (MLCK). Activated MLCKphosphorylates chicken gizzard myosin light chain. If calmodulin isinhibited the rate of myosin light chain phosphorylation is reduced. Totest this, the following experiment is carried out (according to ltoh etal. Biochem. Pharm. 1986,35:217-220). The reaction mixture (0.2 ml)contains 20 mM Tris-HCI (pH 7.5), 0.05 mM [γ-32P] ATP (1 μCi/assaytube), 5 mM MgCI2, 10 μM myosin light chain, 24 nM calmodulin and 0.1 mMCaCI2. MLCK (specific activity: 4.5 moles/min/mg) concentration fromchicken gizzard is 0.1 μg/ml. The incubation is carried out at 30° C.for 4 min. The reaction is terminated by addition of 1 ml of 20%trichloroacetic acid. Then 0.1 ml of bovine serum albumin (1 mg/ml) isadded to the reaction mixture. The sample is then centrifuged for 10min, the pellet is resuspended in 5% trichloroacetic acid. The finalpellet is dissolved in 2 ml of 1 N NaOH and the radioactivity measuredin a liquid scintillation counter. Trypsin-treated MLCK can be preparedas described in ltoh et al. J Pharmacol. Exp. Ther. 1984, 230, p737. Thereaction is initiated by the addition of the ATP and is carried out inthe presence of the potential inhibitors or—as a control—in the presenceof their solvent. Different concentrations of the compounds will betested in the above assay. The concentration of the compound whichresults in 50% decrease of kinase activity will be the IC50concentration.

In some embodiments, a p90S6K and p70S6K inhibitor, rps6, or thespecific Chk2 inhibitors and Cam inhibitors as disclosed herein caninhibit or decrease the activity of the indicated RSK, rps6,Chk2 or Camby at least about 10%, relative to the activity level in the absence ofinhibitors e.g., at least about 15%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, 95%, 99% oreven 100%. In certain embodiments, inhibitors as disclosed herein candecrease expression of the respective protein by about at least 10%, atleast about 15%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, 95%, 99% or even 100%, as comparedto the expression in the absence of the inhibitor.

The expression of p90S6K and p70S6K or Chk2 or Cam or rps6 includes theamount of respective RNA transcribed from a gene, that encodes theprotein, and/or the amount of the amount of protein that is obtained bytranslation of RNA transcribed from a gene. For example, a p90S6K andp70S6K inhibitor as disclosed herein can inhibit expression of p90S6K,p70S6K, Chk2, or Cam or rps6 by at least about 10%, at least about 15%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, 95%, 99% or even 100%, as compared to a referencelevel in the absence of the inhibitor.

Additionally, ability of a compound to inhibit p90S6K and p70S6K, can bealso assessed by measuring a decrease in or an inhibition of biologicalkinase activity as compared to a negative control, e.g. the experimentalcondition in the absence of the inhibitors. Accordingly, a p90S6K andp70S6K inhibitor as disclosed herein can inhibit biological kinaseactivity of p90S6K and p70S6K, by at least about 10%, at least about15%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, 95%, 99% or even 100%, as compared to areference level in the absence of the inhibitor.

In some embodiments, the ability of the inhibitors to inhibit p90S6K orp70S6K, rps6, or Chk2, or Cam, is assessed by rescuing least one of themorphological, hematopoietic or endothelial defects in the Rps29 −/−zebrafish embryo and/or prevent p53 function and nuclear accumulation inA549 lung cancer cell line that have had RPS19 knocked down by siRNA, orreduce p21 levels or increase erythroid markers in CD34+ cells that havehad RPS19 knocked down by siRNA as demonstrated in the Examples herein,as compared to a reference condition without treatment with suchinhibitor.

The dosages of inhibitor to be administered can be determined by one ofordinary skill in the art depending on the clinical severity of thedisease, the age and weight of the patient, the exposure of the patientto conditions that may precipitate outbreaks of psoriasis, and otherpharmacokinetic factors generally understood in the art, such as liverand kidney metabolism. The interrelationship of dosages for animals ofvarious sizes and species and humans based on mg/m³ of surface area isdescribed by E. J. Freireich et al., “Quantitative Comparison ofToxicity of Anticancer Agents in Mouse, Rat, Hamster, Dog, Monkey andMan,” Cancer Chemother. Rep. 50: 219-244 (1966). Adjustments in thedosage regimen can be made to optimize the therapeutic response. Dosescan be divided and administered on a daily basis or the dose can bereduced proportionally depending on the therapeutic situation.

Typically, these drugs will be administered orally, and they can beadministered in conventional pill or liquid form. If administered inpill form, they can be administered in conventional formulations withexcipients, fillers, preservatives, and other typical ingredients usedin pharmaceutical formations in pill form. Typically, the drugs areadministered in a conventional pharmaceutically acceptable formulation,typically including a carrier. Conventional pharmaceutically acceptablecarriers known in the art can include alcohols, e.g., ethyl alcohol,serum proteins, human serum albumin, liposomes, buffers such asphosphates, water, sterile saline or other salts, electrolytes,glycerol, hydroxymethylcellulose, propylene glycol, polyethylene glycol,polyoxyethylenesorbitan, other surface active agents, vegetable oils,and conventional anti-bacterial or anti-fungal agents, such as parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Apharmaceutically-acceptable carrier within the scope of the presentinvention meets industry standards for sterility, isotonicity,stability, and non-pyrogenicity.

The pharmaceutically acceptable formulation can also be in pill, tablet,or lozenge form as is known in the art, and can include excipients orother ingredients for greater stability or acceptability. For thetablets, the excipients can be inert diluents, such as calciumcarbonate, sodium carbonate or bicarbonate, lactose, or calciumphosphate; or binding agents, such as starch, gelatin, or acacia; orlubricating agents such as magnesium stearate, stearic acid, or talc,along with the inhibitor disclosed herein and other ingredients.

The drugs can also be administered in liquid form in conventionalformulations, that can include preservatives, stabilizers, coloring,flavoring, and other generally accepted pharmaceutical ingredients.Typically, when the drugs are administered in liquid form, they will bein aqueous solution. The aqueous solution can contain buffers, and cancontain alcohols such as ethyl alcohol or other pharmaceuticallytolerated compounds.

Alternatively, the drugs can be administered by injection by one ofseveral routes well known in the art. It is, however, generallypreferred to administer the drugs orally.

The drugs can be administered from once per day to up to at least fivetimes per day, depending on the severity of the disease, the totaldosage to be administered, and the judgment of the treating physician.In some cases, the drugs need not be administered on a daily basis, butcan be administered every other day, every third day, or on other suchschedules. However, it is generally preferred to administer the drugsdaily.

In some embodiments, prodrugs of inhibitors disclosed herein also fallwithin the scope of the invention. As used herein, a “prodrug” refers toa compound that can be converted via some chemical or physiologicalprocess (e.g., enzymatic processes and metabolic hydrolysis) to afunctionally active inhibitor (e.g. inhibitors of rps6; p90S6k or p70S6kinhibitors; or the RSk p90 inhibitors SL, or B1; p70s6K inhibitor PF;Cam inhibitor, TF and FLU; Ca2+ Chelator BABTA; chk2 inhibitors CCT andIII, and the inhibitors PerSuc, 221E and ACV; or e.g. the inhibitors ofFIG. 33, or the compounds of Formula I or Formula II.

Thus, the term “prodrug” also refers to a precursor of a biologicallyactive compound that is pharmaceutically acceptable. A prodrug may beinactive when administered to a subject, i.e. an ester, but is convertedin vivo to an active compound, for example, by hydrolysis to the freecarboxylic acid or free hydroxyl. The prodrug compound often offersadvantages of solubility, tissue compatibility or delayed release in anorganism. The term “prodrug” is also meant to include any covalentlybonded carriers, which release the active compound in vivo when suchprodrug is administered to a subject. Prodrugs of an active compound maybe prepared by modifying functional groups present in the activecompound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent active compound. Prodrugsinclude compounds wherein a hydroxy, amino or mercapto group is bondedto any group that, when the prodrug of the active compound isadministered to a subject, cleaves to form a free hydroxy, free amino orfree mercapto group, respectively. Examples of prodrugs include, but arenot limited to, acetate, formate and benzoate derivatives of an alcoholor acetamide, formamide and benzamide derivatives of an amine functionalgroup in the active compound and the like. See Harper, “DrugLatentiation” in Jucker, ed. Progress in Drug Research 4:221-294 (1962);Morozowich et al, “Application of Physical Organic Principles to ProdrugDesign” in E. B. Roche ed. Design of Biopharmaceutical Propertiesthrough Prodrugs and Analogs, APHA Acad. Pharm. Sci. 40 (1977);Bioreversible Carriers in Drug in Drug Design, Theory and Application,E. B. Roche, ed., APHA Acad. Pharm. Sci. (1987); Design of Prodrugs, H.Bundgaard, Elsevier (1985); Wang et al. “Prodrug approaches to theimproved delivery of peptide drug” in Curr. Pharm. Design. 5(4):265-287(1999); Pauletti et al. (1997) Improvement in peptide bioavailability:Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev.27:235-256; Mizen et al. (1998) “The Use of Esters as Prodrugs for OralDelivery of (3-Lactam antibiotics,” Pharm. Biotech. 11, :345-365;Gaignault et al. (1996) “Designing Prodrugs and Bioprecursors I. CarrierProdrugs,” Pract. Med. Chem. 671-696; Asgharnejad, “Improving Oral DrugTransport”, in Transport Processes in Pharmaceutical Systems, G. L.Amidon, P. I. Lee and E. M. Topp, Eds., Marcell Dekker, p. 185-218(2000); Balant et al., “Prodrugs for the improvement of drug absorptionvia different routes of administration”, Eur. J. Drug Metab.Pharmacokinet., 15(2): 143-53 (1990); Balimane and Sinko, “Involvementof multiple transporters in the oral absorption of nucleosideanalogues”, Adv. Drug Delivery Rev., 39(1-3): 183-209 (1999); Browne,“Fosphenytoin (Cerebyx)”, Clin. Neuropharmacol. 20(1): 1-12 (1997);Bundgaard, “Bioreversible derivatization of drugs—principle andapplicability to improve the therapeutic effects of drugs”, Arch. Pharm.Chemi 86(1): 1-39 (1979); Bundgaard H. “Improved drug delivery by theprodrug approach”, Controlled Drug Delivery 17: 179-96 (1987); BundgaardH. “Prodrugs as a means to improve the delivery of peptide drugs”, Arfv.Drug Delivery Rev. 8(1): 1-38 (1992); Fleisher et al. “Improved oraldrug delivery: solubility limitations overcome by the use of prodrugs”,Arfv. Drug Delivery Rev. 19(2): 115-130 (1996); Fleisher et al. “Designof prodrugs for improved gastrointestinal absorption by intestinalenzyme targeting”, Methods Enzymol. 112 (Drug Enzyme Targeting, Pt. A):360-81, (1985); Farquhar D, et al., “Biologically ReversiblePhosphate-Protective Groups”, Pharm. Sci., 72(3): 324-325 (1983);Freeman S, et al., “Bioreversible Protection for the Phospho Group:Chemical Stability and Bioactivation of Di(4-acetoxy-benzyl)Methylphosphonate with Carboxyesterase,” Chem. Soc., Chem. Commun.,875-877 (1991); Friis and Bundgaard, “Prodrugs of phosphates andphosphonates: Novel lipophilic alphaacyloxyalkyl ester derivatives ofphosphate- or phosphonate containing drugs masking the negative chargesof these groups”, Eur. J. Pharm. Sci. 4: 49-59 (1996); Gangwar et al.,“Pro-drug, molecular structure and percutaneous delivery”, Des.Biopharm. Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409-21.(1977); Nathwani and Wood, “Penicillins: a current review of theirclinical pharmacology and therapeutic use”, Drugs 45(6): 866-94 (1993);Sinhababu and Thakker, “Prodrugs of anticancer agents”, Adv. DrugDelivery Rev. 19(2): 241-273 (1996); Stella et al., “Prodrugs. Do theyhave advantages in clinical practice?”, Drugs 29(5): 455-73 (1985); Tanet al. “Development and optimization of anti-HIV nucleoside analogs andprodrugs: A review of their cellular pharmacology, structure-activityrelationships and pharmacokinetics”, Adv. Drug Delivery Rev. 39(1-3):117-151 (1999); Taylor, “Improved passive oral drug delivery viaprodrugs”, Adv. Drug Delivery Rev., 19(2): 131-148 (1996); Valentino andBorchardt, “Prodrug strategies to enhance the intestinal absorption ofpeptides”, Drug Discovery Today 2(4): 148-155 (1997); Wiebe and Knaus,“Concepts for the design of anti-HIV nucleoside prodrugs for treatingcephalic HIV infection”, Adv. Drug Delivery Rev.: 39(1-3):63-80 (1999);Waller et al., “Prodrugs”, Br. J. Clin. Pharmac. 28: 497-507 (1989),content of all of which is herein incorporated by reference in itsentirety.

The inhibitors disclosed herein also include pharmaceutically acceptablesalts thereof. As used herein, the term “pharmaceutically-acceptablesalts” refers to the conventional nontoxic salts or quaternary ammoniumsalts of the inhibitors as disclosed herein, e.g., from non-toxicorganic or inorganic acids. These salts can be prepared in situ in theadministration vehicle or the dosage form manufacturing process, or byseparately reacting an inhibitor in its free base or acid form with asuitable organic or inorganic acid or base, and isolating the salt thusformed during subsequent purification. Conventional nontoxic saltsinclude those derived from inorganic acids such as sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like. See, for example, Berge etal., “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19 (1977), content ofwhich is herein incorporated by reference in its entirety.

In some embodiments of the aspects described herein, representativepharmaceutically acceptable salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,succinate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like.

Treatment of Ribosomal Disorders and Ibosomopathies

In some embodiments, the inhibitors as disclosed herein can be used totreat various disease and disorders associated with ribosomal proteinsor ribosomopathies. For instance, the inhibitors can be used to treat asubject who has a mutation in one or more ribosomal proteins, or have adecreased level of the ribosomal protein.

In some embodiments, the inhibitors as disclosed herein can be used in amethod of treating a subject with a ribosomal disorder such as DiamondBlackfan Anemia (DBA). There are a variety of types of Diamond Blackfananemia, for example, where the subject has DBA1, DBA2, DBA3, DBA4, DBA5,DBA6, DBA7, or DBA8. Diamond Blackfan anemia (DBA), also known asBlackfan-Diamond anemia and Inherited erythroblastopenia, is acongenital erythroid aplasia that usually presents in infancy DBApatients have low red blood cell counts (anemia). The rest of theirblood cells (the platelets and the white blood cells) are normal. Thisis in contrast to Shwachman-Bodian-Diamond syndrome, in which the bonemarrow defect results primarily in neutropenia, and Fanconi anemia,where all cell lines are affected resulting in pancytopenia. A varietyof other congenital abnormalities may also occur. Diamond Blackfananemia is characterized by anemia (low red blood cell counts) withdecreased erythroid progenitors in the bone marrow. This usuallydevelops during the neonatal period. About 47% of affected individualsalso have a variety of congenital abnormalities, including craniofacialmalformations, thumb or upper limb abnormalities, cardiac defects,urogenital malformations, and cleft palate. Low birth weight andgeneralized growth delay are sometimes observed. DBA patients have amodest risk of developing leukemia and other malignancies.

Typically, a diagnosis of DBA is made through a blood count and a bonemarrow biopsy. A diagnosis of DBA is made on the basis of anemia, lowreticulocyte (immature red blood cells) counts, and diminished erythroidprecursors in bone marrow. Features that support a diagnosis of DBAinclude the presence of congenital abnormalities, macrocytosis, elevatedfetal hemoglobin, and elevated adenosine deaminase levels in red bloodcells. Most patients are diagnosed in the first two years of life.However, some mildly affected individuals only receive attention after amore severely affected family member is identified. About 20-25% of DBApatients may be identified with a genetic test for mutations in theRPS19 gene. Approximately 10-25% of DBA cases have a family history ofdisease, and most pedigrees suggest an autosomal dominant mode ofinheritance.

Accordingly, in some embodiments, the inhibitors as disclosed herein canbe used in a method of treating a subject that has a mutation inribosomal protein 19 (RPS19). The phenotype of DBA patients indicates ahematological stem cell defect specifically affecting the erythroidprogenitor population. The RPS19 protein is involved in the productionof ribosomes. Disease features may be related to the nature of RPS19mutations. The disease is characterized by dominant inheritance, andtherefore arises due to a partial loss of RPS19 protein function. I

In alternative embodiments, the inhibitors as disclosed herein can beused in a method of treating a subject with a mutation in ribosomalprotein from at least one of, but not limited to RPS7, RPS10, RPS19,RPS24, PRS26, RPS17, PRS27L RPS29. RPL35A, PRL5 and PPL11. For example,a mutation or variant in RPS19 causes DBA1, and a mutation or variant inRPS24 causes DBA3, a mutation or variant in RPS17 causes DBA4, amutation or variant in RPS34A causes DBA5, a mutation or variant in RPL5causes DBA6, a mutation or variant in RPL11 causes DBA7, and a mutationor variant in RPS7 causes DBA8.

In some embodiments, a subject with a ribosomal disorder has a mutationin a ribosomal protein selected from the group consisting of: rPL2A,rPL2B, rPL3, rpL4A, rPL4B, rPL7A, rPL7B, rPL10, rPL11, rPL16A, rPL17A,rPL17B, rPL18A, rPL18B, Rp119A, rPL19, rPL25, rPL29, rpL31A, rpL31B,rPL36A, rPL40A, rPS1A, rPS6A, rPS6B, rPS14A, rPS15, rPS19, rPS23B,rPS25A, rPS26B, rPS29, rPS29B and rPS31.

In some embodiments of all aspects of the present invention, the methodfurther comprises administering another therapeutic agent to treat theribosomal protein defect, selected from the group consisting of:corticosteroids, blood transfusions and bone marrow transplants andother treatments known to persons of ordinary skill in the art.Corticosteroids can be used to treat anemia in DBA. Blood transfusionscan also be used to treat severe anemia in DBA. Periods of remission mayoccur, during which transfusions and steroid treatments are notrequired. Bone marrow transplantation (BMT) can cure hematologicalaspects of DBA, adverse events in transfusion patients can occur(Diamond Blackfan Anemia Foundation; Pospisilova D et al., (2007).“Successful treatment of a Diamond-Blackfan anemia patient with aminoacid leucine.”. Haematologica 92 (5): e66.)

In some embodiments of all aspects of the present invention, inhibitorsare administered to the subject increases the number of CD71+ erythroidcells in the subject and/or increases hemoglobin levels in the subject.

In some embodiments of all aspects of the present invention, the methodsand inhibitors and as disclosed herein can be used to treat a subjectwith a ribosomal disorder, such as DBA has a symptom of macrocyticanemia and/or craniofacial abnormalities.

In another embodiment, an inhibitor as disclosed herein can be used in amethod of treating a subject with a ribosomal disorder such asmyelodysplasia, for example, but not limited to 5q-myelodysplasia.Myelodysplasia or myelodysplastic syndromes (MDS, formerly known aspreleukemia) are a diverse collection of hematological (blood-related)medical conditions that involve ineffective production (or dysplasia) ofthe myeloid class of blood cells, and where the bone marrow does notfunction normally and produces insufficient number of normal bloodcells.

Patients with MDS often develop severe anemia and require frequent bloodtransfusions. In most cases, the disease worsens and the patientdevelops cytopenias (low blood counts) caused by progressive bone marrowfailure. In about one third of patients with MDS, the disease transformsinto acute myelogenous leukemia (AML), usually within months to a fewyears.

The myelodysplastic syndromes are all disorders of the stem cell in thebone marrow. In MDS, hematopoiesis (blood production) is disorderly andineffective. The number and quality of blood-forming cells declineirreversibly, further impairing blood production.

MDS affects the production of any, and occasionally all, types of bloodcells including red blood cells, platelets, and white blood cells(cytopenias). About 50 percent of pediatric myelodysplasia can beclassified in five types of MDS: refractory anemia, refractory anemiawith ring sideroblasts, refractory anemia with excess blasts, refractoryanemia with excess blasts in transformation, and chronic myelomonocyticleukemia. The remaining 50 percent typically present with isolated orcombined cytopenias such as anemia, leucopenia and/or thrombocytopenia(low platelet count). Although chronic, MDS progresses to become acutemyeloid leukemia (AML) in about 30 percent of patients.

The median age at diagnosis of a MDS is between 60 and 75 years; a fewpatients are younger than 50; MDS diagnoses are rare in children. Malesare slightly more commonly affected than females. Signs and symptoms arenonspecific and generally related to the blood cytopenias include, butare not limited to:

(a) Anemia (low RBC count or reduced hemoglobin)—chronic tiredness,shortness of breath, chilled sensation, sometimes chest pain

(b) Neutropenia (low neutrophil count)—increased susceptibility toinfection

(c) Thrombocytopenia (low platelet count)—increased susceptibility tobleeding and ecchymosis (bruising), as well as subcutaneous hemorrhagingresulting in purpura or petechia[5]

Many individuals are asymptomatic, and blood cytopenia or other problemsare identified as a part of a routine blood count: neutropenia, anemiaand thrombocytopenia (low cell counts of white and red blood cells, andplatelets, respectively); splenomegaly or rarely hepatomegaly; abnormalgranules in cells, abnormal nuclear shape and size; and/or chromosomalabnormalities, including chromosomal translocations and abnormalchromosome number.

Although there is some risk for developing acute myelogenous leukemia,about 50% of deaths occur as a result of bleeding or infection. Leukemiathat occurs as a result of myelodysplasia is notoriously resistant totreatment.

5q-myelodysplasia, (also known as chromosome 5q deletion syndrome,chromosome 5q monosomy, or 5q-syndrome) is a rare disorder caused byloss of part of the long arm (q arm, band 5q31.1) of human chromosome 5.5q-myelodysplasia is characterized by macrocytic anemia oftenthrombocytosis, erythroblastopenia, megakaryocyte hyperplasia withnuclear hypolobation and an isolated interstitial deletion of chromosome5. The 5q-syndrome is found predominantly in females of advanced age.

Some subjects with 5q-myelodysplasia have a decrease in Rps14expression. Deletion of the miR-145 and miR-146 loci has been associatedwith elevated platelet count and megakaryocytic dysplasia associatedwith the 5q-syndrome. 5q-myelodysplasia affects bone marrow cellscausing treatment-resistant anemia and myelodysplastic syndromes thatmay lead to acute myelogenous leukemia. Examination of the bone marrowshows characteristic changes in the megakaryocytes. They are morenumerous than usual, small and mononuclear. There may be accompanyingerythroid hypoplasia in the bone marrow. Accordingly, in someembodiments, a subject with 5q-myelodysplasia can have dysplastic bonemarrow. Subjects with 5q-myelodysplasia can be treated with Lenalidomide(Bennett J et al. (2006). “Lenalidomide in the myelodysplastic syndromewith chromosome 5q deletion”. N. Engl. J. Med. 355 (14): 1456-65; Razaet al., (2008), “Phase 2 study of lenalidomide in transfusion-dependent,low-risk, and intermediate-1 risk myelodysplastic syndromes withkaryotypes other than deletion 5q”. Blood 111 (1): 86-93.)

In some embodiments of all aspects of the present invention, the methodsand inhibitors as disclosed herein can be used to treat a subject with aribosomopathy such as Shwachman-Diamond syndrome, for example, where thesubject has a mutation in Sbds. In some embodiments, a subject withShwachman-Diamond syndrome has one or more symptoms selected frompancreatic insufficiency, bone marrow dysfunction, skeletal deformities.

In another embodiment, an inhibitor as disclosed herein can be used in amethod of treating a subject with a ribosomopathy such as TreacherCollins Syndrome, for example, where the subject has a mutation in TCOF1(nucleolar). Treacher-Collins syndrome is a condition that is passeddown through families (hereditary) that leads to problems with thestructure of the face. Treacher-Collins syndrome is caused by adefective protein called treacle. The condition is passed down throughfamilies (inherited). This condition may vary in severity fromgeneration to generation and from person to person. Symptoms ofTreacher-Collins syndrome include at least one of, but are not limitedto: abnormal or almost completely missing outer part of the ears,hearing loss, very small jaw (micrognathia), very large mouth, defect inthe lower eyelid (coloboma), scalp hair that reaches to the cheeks,cleft palate. Accordingly, a subject with Treacher Collins Syndrome hasone or more craniofacial deformities. While a child with TreacherCollins Syndrome usually will show normal intelligence, diagnosis can bemade on the bases of an examination of the infant which may reveal avariety of problems, including: (a) Abnormal eye shape, (b) Flatcheekbones, (c) Clefts in the face, (d) Small jaw, (e) Low-set ears, (f)Abnormally formed ears, (g) Abnormal ear canal, (h) Hearing loss, (i)Defects in the eye (coloboma that extends into the lower lid), (j)Decreased eyelashes on the lower eyelid, (k) genetic tests can helpidentify gene changes linked to this condition. The diagnosis ofTreacher Collins Syndrome also relies upon clinical and radiographicfindings, and there is a set of typical symptoms within Treacher CollinsSyndrome which can be detected by a critical clinical view. The widespectrum of diseases which have similar characteristics make itsometimes difficult to diagnose TCS. The OMENS classification wasdeveloped as a comprehensive and stage-based approach to differentiatethe diseases. This acronym describes five distinct dysmorphicmanifestations, namely O; orbital asymmetry, M; mandibular hypoplasia,E; auricular deformity, N; nerve development and S; soft-tissue disease.

Selection of Subjects for Administration with a PharmaceuticalComposition Comprising the Inhibitor

In some embodiments, a subject amenable or suitable for treatment with acomposition comprising an inhibitor as disclosed herein can be selectedbased on decreased levels of hematopoietic cells and decreased flklexpression in CD34+ cells, as compared to a control reference normallevels of hematapoeitc cells and flkl expression level from a normalsubject. Additionally, a subject amenable or suitable for treatment witha composition comprising an inhibitor as disclosed herein can beselected based on increased levels of p21 expression in CD34+ cells ascompared to a control reference p21 expression level. In someembodiments, a subject amenable or suitable for treatment with acomposition comprising an inhibitor as disclosed herein can be selectedbased on decreased CD71+ expression and decreased glycophorin A (GPA)expression in CD34+ cells as compared to a control reference CD71+ andGPA expression level, e.g., in a sample from a normal subject not havinga ribosomal disorder or ribosomopathy. In some embodiments, the normalreference levels are the based on the level of hematopoietic cells, flklexpression, CD71+ expression, GPA expression, p21 expression levels in asample from a normal subject not having a ribosomal disorder orribosomopathy, or a control cell line, or cells from a normal tissuesample, where in the tissue sample is a biological tissue sample from atissue matched, and species matched and age matched biological sample.

In some embodiments, the levels of flk 1 expression, CD71+ expression,GPA expression, and p21 expression levels are measured in a biologicalsample comprising hematopoietic cells or erythroid cells or erythroiddifferentiated cells. In some embodiments, a biological sample obtainedfrom the subject comprises cancer cells, and can be a biological samplewhich is serum plasma, blood or tissue sample. In alternativeembodiments, the biological sample includes, for example blood, plasma,serum, urine, spinal fluid, plural fluid, nipple aspirates, lymph fluid,external secretions of the skin, respiratory, internal andgenitoururinary tracts, bile, tears, sweat, saliva, organs, milk cellsand primary ascite cells, biopsy tissue sample, an in vitro or ex vivocultivated biopsy tissue sample.

Pharmaceutical Compositions Comprising the Inhibitor

Another aspect of the present invention relates to pharmaceuticalcompositions for treatment of diseases or disorders associated withribosomal proteins or dysfunction or where a subject has aribosomopathy, e.g., DBA, myelodysplasia, for example, but not limitedto 5q-myelodysplasia, Shwachman-Diamond syndrome and Treacher CollinsSyndrome. In some embodiments, a pharmaceutical composition of theinvention comprises a therapeutically effective amount of at least oneof the inhibitors as disclosed herein. In one embodiment, the inhibitoris, for example, but not limited to, inhibitors of rps6; the RSk p90inhibitors SL, or B1; p70s6K inhibitor PF; Cam inhibitor, TF and FLU;Ca2+ Chelator BABTA; chk2 inhibitors CCT and III, and the inhibitorsPerSuc, 221E and ACV; or e.g. the inhibitors of FIG. 33, or thecompounds of Formula I or Formula II.

An inhibitor as disclosed herein can be used in an amount of about 0.001to 10 mg/kg of body weight or about 0.005 to 8 mg/kg of body weight orabout 0.01 to 6 mg/kg of body weight or about 0.1 to 0.2 mg/kg of bodyweight or about 1 to 2 mg/kg of body weight. In some embodiments, aninhibitor can be used in an amount of about 0.1 to 1000 μg/kg of bodyweight or about 1 to 100 μg/kg of body weight or about 10 to 50 μg/kg ofbody weight. In some embodiments, the inhibitor as disclosed herein canbe used at a concentration of about 0.001mg/ml or 0.1mg/ml or a higherconcentration of 0.1mg/ml. In some embodiments, a pharmaceuticalcomposition comprises at least one inhibitor at a concentration of about0.01 μM to 300 μM, or about 0.1 μM to 150 μM, or about 1 μM to 50 μM, orabout 1 μM to 25 μM. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.

Depending on routes of administration, one of skill in the art candetermine and adjust an effective dosage of an inhibitor disclosedherein to a subject such as a human subject accordingly, by determiningpharmacokinetics and bioavailability of an inhibitor and analyzingdose-response relationship specific to an inhibitor in animal modelssuch as a mouse.

Toxicity and therapeutic efficacy can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compositions that exhibit large therapeutic indices, are preferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. Thetherapeutically effective dose can be determined by one of ordinaryskill in the art, e.g. using cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe therapeutic which achieves a half-maximal inhibition of symptoms) asdetermined in cell culture by methods disclosed in the Examples. Aneffective dose of the inhibitor can be determined in an animal model bymeasuring the levels of hemoglobin over the course of treatment with theinhibitor as compared to no treatment. In some embodiments, a dosagecomprising the inhibitor is considered to be effective if the dosageincreases hemoglobin levels, red cell number, and/or reduces expressionof p21 in CD34+ cells by at least about 15%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, 95%,99% or even 100%, as compared to a control (e.g. in the absence of theinhibitor),In some embodiments, a therapeutically effective amount ofthe inhibitor administered to a subject is dependent upon factors knownto a person of ordinary skill, including bioactivity and bioavailabilityof the inhibitor (e.g. half-life and stability of the inhibitor in thebody), chemical properties of the inhibitor (e.g. molecular weight,hydrophobicity and solubility); route and frequency of administration,time of administration (e.g. before or after a meal), and the like.Further, it will be understood that the specific dose of thepharmaceutical composition comprising the inhibitor as disclosed hereinto provide the ttherapeutic or prophylactic benefits can depend on avariety of factors including physical condition of the subject (e.g.age, gender, weight), medical history of the subject (e.g. medicationsbeing taken, other diseases or disorders) and clinical condition of thesubject (e.g. health condition, stage of the disease). The precise doseof a pharmaceutical composition comprising the inhibitor can bedetermined by methods known to a skilled artisan such as pharmacologistsand physicians.

According to the invention, an inhibitor as disclosed herein can beadministered prophylactically or therapeutically to a subject prior to,simultaneously or sequentially with other therapeutic regimens or agents(e. g. multiple drug regimens), in a therapeutically effective amount.In some embodiments, the inhibitor is administered concurrently withother therapeutic agents can be administered in the same or differentcompositions. Additional therapeutic agents or regimens include, but arenot limited to, steroids, corticosteroids, blood transfusions and bonemarrow transplants.

The active ingredients (e.g. inhibitors of p90S6K; inhibitors of p60S6K;RSk p90 inhibitors SL, or B1, or Sk; p70s6K inhibitor PF; Cam inhibitor,TF or FLU; Ca2+ Chelator BABTA; chk2 inhibitors, CCT or III, and theinhibitors PerSuc, or 221E or ACV; or e.g. the inhibitors of FIG. 33, orthe compounds of Formula I and Formula II.) of the pharmaceuticalcomposition according to the invention can be administered to anindividual by any route known to persons skilled in the art. The routesof administration include intradermal, transdermal (e.g. in slow releaseformulations), intramuscular, intraperitoneal, intravenous,subcutaneous, oral, buccal, nasal, rectal, epidural, topical,intrathecal, rectal, intracranial, intratracheal and intrathecal andintranasal routes. Any other therapeutically efficacious route ofadministration can be used, for example absorption through epithelial orendothelial tissues or systemic administration. In addition, aninhibitor according to the invention can be administered together withother components of biologically active agents such as pharmaceuticallyacceptable surfactants, excipients, carriers, diluents and vehicles.

For parenteral (e.g. intravenous, subcutaneous, intramuscular)administration, an inhibitor can be formulated as a solution,suspension, emulsion or lyophilized powder in association with apharmaceutically acceptable parenteral vehicle (e.g. water, saline,dextrose solution) and additives that maintain isotonicity (e g.mannitol) or chemical stability (e.g. preservatives and buffers). Theformulation is sterilized by commonly used techniques.

In some embodiments, the route of administration is administration bysubcutaneous route. Intramuscular administration is another alternativeroute of administration. In some embodiments, a pharmaceuticalcomposition comprising an inhibitor can be administered as a formulationadapted for systemic delivery. In some embodiments, the compositions canbe administered as a formulation adapted for delivery to specificorgans, for example but not limited to the liver. In some embodiments, apharmaceutical composition comprising an inhibitor as disclosed hereincan be administered as a formulation adapted not to pass through theblood-brain barrier.

Alternatively, in some embodiments, a pharmaceutical composition can beincorporated in a gel, sponge, or other permeable matrix (e.g., formedas pellets or a disk) and placed in proximity to the liver endotheliumfor sustained, local release. The composition comprising an inhibitorcan be administered in a single dose or in multiple doses, which areadministered at different times.

The exact route of administration as well as the optimal dosages can bedetermined by standard clinical techniques for each specific case,mainly based on the nature of the disease or disorder and on the stageof this disease. Preferably, the medicament according to the presentinvention is applied locally or systemically, in particular, orally,intravenously, parenterally, epicutaneously, subcutaneously,intrapulmonarily by inhalation or bronchoalveolar lavage,intramuscularily, intracranially, locally into intervertebral discs orother connective tissues.

As disclosed herein, a pharmaceutical composition comprising aneffective amount of at least one inhibitor can be administered to asubject for the therapeutic treatment or prevention (e.g. prophylactictreatment) of ribosomal diseases and disorders or ribosomopathies.

In some embodiments, a composition of the invention comprising aninhibitor as disclosed herein is formulated for ribosomal diseasesand/or ribosomophaties, e.g. DBA, myelodysplasia, for example, but notlimited to 5q-myelodysplasia, Shwachmanp-Diamond syndrome and TreacherCollins Syndrome. In one embodiment, an inhibitor as disclosed herein isa derivative, analogue, prodrug, or pharmaceutically acceptable saltsthereof.

In some embodiments, a pharmaceutical composition comprising at leastone of the inhibitors disclosed herein further comprises a secondtherapeutic agent. In one embodiment, the second therapeutic agent is acorticosteroid. In some embodiments, the second therapeutic agent is acalcium channel blocker, as disclosed herein.

In prophylactic applications, pharmaceutical compositions (ormedicaments) comprising an inhibitor can be administered to a subjectsusceptible to, or otherwise at risk of, a ribosomal disease or disorderand/or ribosomopathy in an amount sufficient to eliminate or reduce therisk or delay the onset of the disease. In one embodiment, apharmaceutical composition of the invention disclosed herein comprises ainhibitor of rps6; a RSk p90 inhibitor e.g. SL, or B1; or a p70s6Kinhibitor, e.g. PF; or the Cam inhibitor, TF or FLU; or the Ca2+Chelator BABTA; or the chk2 inhibitor, CCT or III; or one of theinhibitors PerSuc, 221E, or ACV, DB-4-088-2 (088-2); DB-4-088-3 (088-3);DB-4-086 (086); DB-4-087-2 (087-2); DB-4-087-3 (087-3); DB-4-089 (089),or a compound of Formual I or Formula II; or rps6 inhibitor; orenantiomers, prodrugs, derivatives or pharmaceutically acceptable saltsthereof

In therapeutic applications, according to the invention provided herein,when an effective amount or effective dose of a pharmaceuticalcomposition comprising an inhibitor as disclosed herein can beadministered to the subject with a ribosomal disease or disorder and/orribosomopathy so that at least one of the symptoms of such a ribosomaldisease can be delayed or inhibited. In some embodiments, administrationof an effective amount or effective dose of a pharmaceutical compositioncomprising an inhibitor to a subject with a ribosomal disease ordisorder and/or ribosomopathy can inhibit or delay progression of facialabnormalities, and/or other symptoms associated with the ribosomaldisease or ribosomopathy. In further embodiments, treating subjects withan effective dose of a pharmaceutical composition comprising aninhibitor can prevent or delay a symptom of the ribosomal disease orribosomopathy in the subject.

In some embodiments, the present invention also provides compositionscomprising an inhibitor as discussed herein for practicing thetherapeutic and prophylactic methods described herein. In someembodiments, combinations of an inhibitor and another therapeutic agentcan be tailored to be combined in a pharmaceutical composition, whereeach therapeutic can target a different symptom, a different disease ora different disorder. In further embodiments, the inhibitor and anothertherapeutic can be mixed together in a pharmaceutical composition asdisclosed herein. In other embodiments, an inhibitor and anothertherapeutic can be present in a different formulation when combined in apharmaceutical composition. For example, in one embodiment, theinhibitor can be present in a liquid formulation, while anothertherapeutic can be lyophilized into powder. The formulations ofdifferent active ingredients in a pharmaceutical composition asdisclosed herein (e.g. inhibitors of rps6; Inhibitors of p90S6K orp70S6K; RSk p90 inhibitors SL, B1; p70s6K inhibitor PF; Cam inhibitor,TF and FLU; Ca2+ Chelator BABTA; chk2 inhibitors CCT and III, and theinhibitors PerSuc, 221E and ACV; or the inhibitor compounds of FIG. 33,or the compounds of Formula I or II.) can be optimized accordingly byvarious factors such as physical and chemical properties of a drug,bioavailability, route of administration, and whether it is a sustainedor a burst release for the drug. Therapeutic and prophylacticcompositions of the present invention can further comprise aphysiologically tolerable carrier together with an inhibitor asdisclosed herein (inhibitors of rps6; inhibitors of p90S6K or p70S6K;RSk p90 inhibitors SL, B1; p70s6K inhibitor PF; Cam inhibitor, TF andFLU; Ca2+ Chelator BABTA; chk2 inhibitors CCT and III, and theinhibitors PerSuc, 221E and ACV, or the compounds of Formula I orFormula II), or derivatives, enantiomers, prodrugs or pharmaceuticallyacceptable salts thereof. In additional embodiments, an inhibitor andanother therapeutic can employ different physiologically tolerablecarriers when combined in a pharmaceutical composition of the inventionas disclosed herein.

In some embodiments, a pharmaceutical composition as disclosed hereincomprises an inhibitor together with other therapeutics and apharmaceutically acceptable excipient. Suitable carriers for aninhibitor of the invention, and their formulations, are described inRemington's Pharmaceutical Sciences, 16th ed., 1980, Mack PublishingCo., edited by Oslo et al. Typically an appropriate amount of apharmaceutically acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the carrier include buffers suchas saline, Ringer's solution and dextrose solution. Further carriersinclude sustained release preparations such as semipermeable matrices ofsolid hydrophobic polymers, which matrices are in the form of shapedarticles, e.g. liposomes, films or microparticles. It will be apparentto those of skill in the art that certain carriers can be morepreferable depending upon for instance the route of administration andconcentration of the inhibitor being administered.

In some embodiments, bioavailability of the inhibitor according to theinvention can also be improved by using conjugation procedures whichincrease the half-life of the inhibitor in a subject, for examplelinking the inhibitor to polyethylene glycol, as described in WO92/13095, which is incorporated herein in its entirety by reference.

In some embodiments, bioavailability of the inhibitor according to theinvention can be also enhanced by encapsulating a the inhibitor inbiocompatible delivery vehicles which increase the half-life of aninhibitor in a human body. Exemplary biocompatible delivery vehiclesinclude polymeric vehicles such as PEG-based vehicles, or liposome-basedvehicles.

In some embodiments, the inhibitor can be dissolved or dispersed as anactive ingredient in the physiologically tolerable carrier to increasethe half-life of the inhibitor in a subject.

The preparation of a pharmacological composition that contains activeingredients (e.g inhibitors of rps6; inhibitors of p90S6K or p70S6K; RSkp90 inhibitors SL, or B1; p70s6K inhibitor PF; Cam inhibitor, TF andFLU; Ca2+ Chelator BABTA; chk2 inhibitors CCT and III, and theinhibitors PerSuc, 221E and ACV; or e.g. the inhibitors of FIG. 33; orthe compounds of Formulas I or II) dissolved or dispersed therein iswell understood in the art and need not be limited based on formulation.Typically such compositions are prepared as injectable either as liquidsolutions or suspensions, however, solid forms suitable for solution orsuspension in liquid prior to use can also be prepared. The preparationcan also be emulsified or presented as a liposome composition. In someembodiments, the inhibitor can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. In addition, if desired, the composition comprising theinhibitor can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents and the like whichenhance the effectiveness of the active ingredient.

Physiologically tolerable carriers (i.e. physiologically acceptablecarriers) are well known in the art. Selection of pharmaceuticallyacceptable carriers can be accomplished by means of administration by askilled artisan. For example, if the composition is orally administered,it can be formulated in coated tablets, liquids, caplets and so forth.Exemplary of liquid carriers are sterile aqueous solutions that containno materials in addition to the active ingredients and water, or containa buffer such as sodium phosphate at physiological pH value,physiological saline or both, such as phosphate-buffered saline. Stillfurther, aqueous carriers can contain more than one buffer salt, as wellas salts such as sodium and potassium chlorides, dextrose, polyethyleneglycol and other solutes. For topical application, the carrier may be inthe form of, for example, and not by way of limitation, an ointment,cream, gel, paste, foam, aerosol, suppository, pad or gelled stick. Insome embodiments, compositions are prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared. The preparation also can be emulsified or encapsulated inliposomes or micro particles such as polylactide, polyglycolide, orcopolymer for enhanced adjuvant effect, as discussed above (see Langer,Science 249, 1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28,97-119 (1997). An inhibitor as disclosed herein can be administered inthe form of a depot injection or implant preparation which can beformulated in such a manner as to permit a sustained or pulsatilerelease of the active ingredient.

As used herein, the ‘inhibitors as disclosed herein’ include e.g.inhibitors of rps6; ihibitors of p90S6K or p70S6K; RSk p90 inhibitorsSL, or B1; p70s6K inhibitor PF; Cam inhibitor, TF and fluphenazine(FLU); the Ca2+ Chelator BABTA; chk2 inhibitors CCT and III, and theinhibitors PerSuc, 221E and ACV;and DB-4-083, DB-4-084, DB-4-088-2,DB-4-088-3,DB-4-086, DB-4-087-2, DB-4-087-3, DB-4-089, The compounds ofFormula I and Formula II, and derivatives and analogs thereof (See e.g.FIG. 5, FIG. 31, FIG. 32, and FIG. 33, for structures).

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications. For suppositories, binders and carriersinclude, for example, polyalkylene glycols or triglycerides; suchsuppositories can be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1%-2%. Oralformulations include excipients, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25%-70%.

A skilled artisan will be able to determine the appropriate way ofadministering pharmaceutical compositions comprising at least one LSFinhibitor as disclosed herein in view of the general knowledge and skillin the art.

Treatment Regimes

Another aspect of the present invention relates to methods fortherapeutic and prophylactic treatment of diseases or disorders, whereinhibition of p53 activation is desirable for the treatment orprevention of a ribosomal disorder or a ribosomopathy. The methodscomprise administering to a subject in need thereof a pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone inhibitor selected from for example, any, or a combination, ofcompounds such as small molecule or protein inhibitors of rps6, p90S6Kor p70S6K; the p90S6K inhibitors SL and B1; the p70s6K inhibitor PF; theCam inhibitor, TF and FLU; the Ca2+chelator BABTA; the chk2 inhibitorsCCT and III, and the inhibitors PerSuc, 221E and ACV; and those of FIG.33, and of Formula I and Formula II), and analogues and variants asdisclosed herein.

In one embodiment, Diamond-Blackfan anemia (DBA) is treated or preventedby the methods and compositions of the present invention with aninhibitor as disclosed herein.

Effective doses of the pharmaceutical composition comprising aninhibitor as disclosed herein, for the treatment of ribosome proteindiseases or disorders or associated with a ribosomopathy depend uponmany different factors, including means of administration, physiologicalstate of the subject, whether the subject is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Depending on the clinical condition of a subject, dosageand frequency of pharmaceutical compositions of the present inventioncan be adjusted accordingly over time by one of the skill in the art,e.g. physicians.

In therapeutic applications, a relatively high dosage in relativelyshort intervals is sometimes required until progression of the diseaseis reduced or terminated, or until the subject shows partial or completeamelioration of symptoms of disease. Thereafter, the subject can beadministered a prophylactic regime. For example, subjects with DBA canbe treated with an inhibitor as disclosed herein at an effective dose ina therapeutic regimen accordingly to decrease the p21 levels and or p53levels back to a normal level, and then be administered a maintenancedose, e.g., prophylactically. In some embodiments, an inhibitor asdisclosed herein can be administered to subjects prior to, concurrentlywith, or sequentially to treatment with a corticosteroid, and/or whenthe subject us undergoing an adjuvant therapy, such as a bloodtransfusion and/or bone marrow transplant. In some embodiments forexample, a DBA subject which is selected for other therapeuticprocedures or surgeries, such as blood transfusions and/or bone marrowtransplant, can be subjected to a treatment with an inhibitor asdisclosed herein. For example, a pharmaceutical composition of theinvention can be administered prior to, during or after therapeuticprocedures. Route of administration can vary with therapeutic proceduresor surgeries and can be determined by a skilled artisan. In yet anotherembodiment, compositions and methods of the invention can be used as anadjuvant therapy.

In some embodiments, the subject is a human, and in alternativeembodiments the subject is a non-human mammal. Treatment dosages need tobe titrated to optimize safety and efficacy. The amount of an inhibitordepends on the stage of the disease, as well as the species.

In some embodiments, an inhibitor can be administered to a subject in apharmaceutical composition comprising an amount of an inhibitor of about0.001 to 10 mg/kg of body weight or about 0.005 to 8 mg/kg of bodyweight or about 0.01 to 6 mg/kg of body weight or about 0.1 to 0.2 mg/kgof body weight or about 1 to 2 mg/kg of body weight. In someembodiments, an inhibitor can be used in an amount of about 0.1 to 1000μg/kg of body weight or about 1 to 100 μg/kg of body weight or about 10to 50 μg/kg of body weight. In some embodiments, an inhibitor can beadministered at a concentration of about 0.001mg/ml or 0.1mg/ml or ahigher concentration of 0.1mg/ml. In alternative embodiments, apharmaceutical composition comprises at least one inhibitor at aconcentration of about 0.01 μM to 300 μM, or about 0.1 μM to 150 μM, orabout 1 μM to 50 μM, or about 1 μM to 25 μM.

In some embodiments, an inhibitor as disclosed herein can beadministered to a subject according to the methods in an effective doseto increase the levels of CD71+ cells in an erythroid cell populationobtained from the subject by at least about 1%, at least about 2%, atleast about 3%, at least about 5%, at least about 10%, at least about15%, least about 20%, at least about 30%, at least about 40%, at leastabout 50%, or more than 50%, as compared to in the absence of theinhibitor.

In another embodiment, an inhibitor as disclosed herein can beadministered to a subject according to the methods as disclosed hereinin an effective dose to decrease the levels of p21 expression in CD34+cells present in an erythroid cell population obtained from the subjectby at least about 1%, at least about 2%, at least about 3%, at leastabout 5%, at least about 10%, at least about 15%, least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%,at least about 98%, at least about 99%, or more than99%, as compared to in the absence of the inhibitor.

Generally, effective dosages and dosing schedules can be adjusted basedon, for example, the outcome of the treatment such as whether thesubject has reduced symptoms of anemia, and/or whether at least one ofthe symptoms associated with the ribosomal protein disorder, such as DBAis reduced. In accordance with the teachings provided herein, theeffectiveness of the treatment can be monitored by obtaining abiological sample from a subject, e.g. a blood serum sample, anddetermining the level of biomarkers for DBA, such as percentage of CD71+cells in a erythroid cell population and/or level of p21 in CD34+ cells,using methods well known in the art and the diagnostic methods asdisclosed later herein.

In some embodiments, the daily dose administered to a subject in a formof a bolus composition comprising an inhibitor can be given in a singledose, in divided doses or in sustained release form effective to obtainthe desired results. Second or subsequent administrations can beperformed at a dosage which is the same, less than or greater than theinitial or previous dose administered to the individual. A second orsubsequent administration can be administered during or prior to onsetof the disease. It is also within the skill of the art to start doses atlevels lower than required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect is achieved.

The pharmaceutical compositions comprising at least one inhibitor asdisclosed herein can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraperitoneal, intranasal orintramuscular means for prophylactic and/or therapeutic treatment. Forexample, for treatment of cancer, e.g., HCC, a pharmaceuticalcomposition comprising at least one LSF inhibitor can be injectedsystemically such as by intravenous injection, or by injection orapplication to the relevant site, such as by direct injection into atumor, or direct application to the site when the site is exposed insurgery. Other routes of administration of an inhibitor as disclosedherein are intramuscular (i.m.), intravenous (i.v.), subcutaneous(s.c.), or orally, although other routes can be equally effective.Intramuscular injection is most typically performed in the arm or legmuscles. In some methods, an inhibitor as disclosed herein can beadministered as a sustained release composition or device, such as aMedipad™ device.

In some embodiments, an inhibitor as disclosed herein can optionally beadministered in combination with other agents that are at least partlyeffective in treatment of ribosomal protein diseases and disorders, suchas blood transfusions, bone marrow transplants and the like. In otherembodiments, an inhibitor of the invention can be administered prior to,concurrently, or after administration of another therapeutics thattargets another disease or disorder, or a different symptom.

In various embodiments, an inhibitor can be a pro-drug, where it isactivated by a second agent. Accordingly, in such embodiments,administration of such the second agent which activates the pro-drug ofthe inhibitor into its active form can be administered the same time,concurrent with, or prior to, or after the administration of thepharmaceutical composition comprising an inhibitor as disclosed herein.

In some embodiments, an inhibitor as disclosed herein is oftenadministered as pharmaceutical compositions comprising an activetherapeutic agent, e.g. inhibitors of rps6; inhibitors of p90S6K orp70S6K; RSk p90 inhibitors SL and B1; p70s6K inhibitor PF; Caminhibitor, TF and FLU; Ca2+ Chelator BABTA; chk2 inhibitors CCT and III,and the inhibitors PerSuc, 221E and ACV;and those of FIG. 33, and ofFormulas I and II), and a variety of other pharmaceutically acceptablecomponents. See Remington's Pharmaceutical Science (15th ed., MackPublishing Company, Easton, Pa., 1980). The formulation of thecompositions depends on the intended mode of administration andtherapeutic application. The compositions can also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, non-therapeutic, non-immunogenic stabilizers and the like.However, some reagents suitable for administration to animals may notnecessarily be used in compositions for human use.

For parenteral administration, an inhibitor as disclosed herein can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier which can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Other mode of administration includes systemic delivery. In someembodiments, at least one inhibitor as disclosed herein can be injectedsystemically such as by intravenous injection, or by injection orapplication to the relevant site, such as direct application to the sitewhen the site is exposed in surgery. In some embodiments, apharmaceutical composition of the invention can be formulated in atablet and used orally for systemic administration. In variousembodiments, pharmaceutical compositions of the invention can furthercomprises non-active ingredients (i.e. ingredients that have notherapeutic values for treatment of diseases, disorders or symptoms),such as physiologically acceptable carriers.

In various embodiments, modification of an inhibitor by addition of apolymer is specifically contemplated, for example, using a covalentattachment to a polymer. In other embodiments, an inhibitor can be mixedwith or encapsulated in a biocompatible polymer.

In another aspect, biodegradable or absorbable polymers can provideextended, often localized, release of an inhibitor as disclosed herein.The potential benefits of an increased half-life or extended release fora therapeutic agent are clear. A potential benefit of localized releaseis the ability to achieve much higher localized dosages orconcentrations, for greater lengths of time, relative to broadersystemic administration, with the potential to avoid possibleundesirable side effects that may occur with systemic administration.

Bioabsorbable polymeric matrix suitable for delivery of an inhibitor asdisclosed herein, or variants or fragments or derivatives thereof can beselected from a variety of synthetic bioabsorbable polymers, which aredescribed extensively in the literature. Such synthetic bioabsorbable,biocompatible polymers, which may release proteins over several weeks ormonths can include, for example, poly-hydroxy acids (e.g. polylactides,polyglycolides and their copolymers), polyanhydrides, polyorthoesters,segmented block copolymers of polyethylene glycol and polybutyleneterephtalate (POLYACTIVE™), tyrosine derivative polymers orpoly(ester-amides). Suitable bioabsorbable polymers to be used inmanufacturing of drug delivery materials and implants are discussed e.g.in U.S. Pat. Nos. 4,968,317, 5,618,563 (which are incorporated herein intheir entirety by reference), among others, and in “Biomedical Polymers”edited by S. W. Shalaby, Carl Hanser Verlag, Munich, Vienna, New York,1994 and in many references cited in the above publications. Theparticular bioabsorbable polymer that should be selected will dependupon the particular patient that is being treated.

The methods of the present invention also are useful for monitoring acourse of treatment being administered to a subject. The methods can beused to monitor both therapeutic treatment on symptomatic subject andprophylactic treatment on asymptomatic subject.

A treatment administered to a subject is considered to be effective ifthe level of expression of p21 in CD34+ cells present in a biologicalsample obtained from the subject is decreased by at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 98%, about 99% or about 100% as comparedto a reference level, or in the absence of the inhibitor. In suchembodiments, the reference level is the measurement of p21 in CD34+cells present in a biological sample obtained from the subject at aprevious time point, e.g., who has not been administered the inhibitors.Based on the outcome of treatment, the dosage and frequency ofadministration using the methods and compositions as disclosed hereincan be adjusted accordingly by one of skill in the art.

One can use any immunoassay to determine the level of p21 expression inCD34+ cells in a biological sample, such as ELISA or immunohistochemicalmethods which are commonly known in the art and are encompassed for usein the present invention.

Kits

Another aspect of the present invention relates to a kit comprising oneor more inhibitors as disclosed herein (e.g. inhibitors of rps6;inhibitors of p90S6K or p70S6K; RSk p90 inhibitors SL, or B1; p70s6Kinhibitor PF; Cam inhibitor, TF and FLU; Ca2+ Chelator BABTA; chk2inhibitors CCT and III, and the inhibitors PerSuc, 221E and ACV; and thecompounds of FIG. 33, and of Formulas I and II), and instructions forcarrying out a method as disclosed herein.

In some embodiments, a kit can optionally additionally comprise reagentsor agents for measuring the level of p21 expression in a biologicalsample from the subject, such as, for example, a blood sample, forexample to identify the efficacy of treatment with the inhibitor asdisclosed herein. Such agents are well known in the art, and includewithout limitation, labeled antibodies that specifically bind to p21protein and/or mRNA and the like. In some embodiments, the labeledantibodies are fluorescently labeled, or labeled with magnetic beads andthe like. In some embodiments, a kit as disclosed herein can furthercomprise at least one or more reagents for profiling and annotating abiological sample from the subject in high throughput assay.

In some embodiments, the kit can further comprise instructions foradministering a composition comprising an inhibitor to a subject in needthereof, e.g., with a ribosomal protein disease or disorder, e.g., DBAand instructions for doses and the like.

In addition to the above mentioned component(s), the kit can alsoinclude informational material. The informational material can bedescriptive, instructional, marketing or other material that relates tothe methods described herein and/or the use of the components for theassays, methods and systems described herein.

In some embodiments, the methods and kits comprising an inhibitor asdisclosed herein can be performed by a service provider, for example,where an investigator or physician can send the biological sample to adiagnostic laboratory service provider to measure the level of p21expression in CD34+ cells, and/or the level of CD71+ cells in aerythroid cell population present in the biological subject from thesubject. In such an embodiment, after performing the such measurements,the service provider can provide the investigator or physician a reportof the efficacy of the inhibitor and/or report if the subject is asuitable or amenable to be treated with an inhibitor according to themethods and composition as disclosed herein.

In alternative embodiments, a service provider can provide theinvestigator with the raw data of the levels of p21 p53 expression inCD34+ cells, and/or the levels of CD71+ cells in a erythroid cellpopulation present in the biological subject from the subject and leavethe analysis to be performed by the investigator or physician. In someembodiments, the report is communicated or sent to the investigator viaelectronic means, e.g., uploaded on a secure web-site, or sent viae-mail or other electronic communication means. In some embodiments, theinvestigator can send the samples to the service provider via any means,e.g., via mail, express mail, etc., or alternatively, the serviceprovider can provide a service to collect the samples from theinvestigator and transport them to the diagnostic laboratories of theservice provider. In some embodiments, the investigator can deposit thesamples to be analyzed at the location of the service providerdiagnostic laboratories. In alternative embodiments, the serviceprovider provides a stop-by service, where the service provider sendpersonnel to the laboratories of the investigator and also provides thekits, apparatus, and reagents for performing the assays to measure thelevels of p21 expression in CD34+ cells, and/or the level of CD71+ cellsin a erythroid cell population present in the biological subject fromthe subject as disclosed herein in the investigators laboratories, andanalyses the result and provides a report to the investigator for eachsubject, and leaves the physician to make appropriate recommendations oftreatment, and dose to administer the subject with a compositioncomprising an inhibitor according to the methods as disclosed herein.

The following numbered paragraphs represent embodiments of theinvention:

Paragraph 1 A method of treating a subject with a ribosomal disorder orribosomopathy, comprising administering an effective amount of aninhibitor of ribosomal s6 kinase, RSK (p90S6k), to the subject todecrease p90S6K activity and decrease active p53 in at least one ofCD34+ cells, erythroid cells or erythroid differentiated cells in thesubject.

Paragraph 2, the method of paragraph 1, wherein the inhibitor of p90S6kinhibits a variant selected from the group consisting of RSK1, RSK2 andRSK3.

Paragraph 3, the method of paragraph 1, wherein the inhibitor of p90S6kselectively inhibits RSK2.

Paragraph 4, the method of paragraph 1, wherein the inhibitor of p90S6kis a nucleic acid, a small molecule compound, or a protein.

Paragraph 5, the method of paragraph 1, wherein the inhibitor of p90S6kis SL0101 (SL), or a derivative or analogue of SL0101 (SL), whereinSL0101 (SL) has the following structure:

Paragraph 6, the method of paragraph 1, wherein the inhibitor of p90S6kis BI-D1870 (BI) or a derivative or analogue of BI-D1870 (BI), whereinBI-D1870 (BI) has the following structure:

Paragraph 7, a method of treating a subject with a ribosomal disorder orribosomopathy, comprising administering an effective amount of aninhibitor of RSK (p70S6K) to the subject to decrease p70S6K activity anddecrease active p53 in at least one of CD34+ cells, erythroid cells orerythroid differentiated cells in the subject.

Paragraph 8, the method of paragraph 7, wherein the inhibitor of p70S6kis a nucleic acid, small molecule compound, or a protein.

Paragraph 9, the method of paragraph 7, wherein the inhibitor of p70S6kis PF-4708671 (PF), or a derivative or analogue of PF-4708671 (PF),wherein PF-4708671 (PF) has the following structure:

Paragraph 10, a method of treating a subject with a ribosomal disorderor ribosomopathy, comprising administering an effective amount of aninhibitor of Chk2 to the subject to decrease active p53 in at least oneof CD34+ cells, erythroid cells or erythroid differentiated cells in thesubject, wherein the inhibitor of Chk2 comprises a compound selectedfrom the group consisting of CCT and III, or derivatives thereof,wherein CCT and III have the following structures:

Paragraph 11, a method of treating a subject with a ribosomal disorderor ribosomopathy, comprising administering an effective amount of aninhibitor of calmodulin to the subject to decrease active p53 in atleast one of CD34+ cells, erythroid cells or erythroid differentiatedcells in the subject, wherein the inhibitor of calmodulin is aphenothiazine compound, or a derivative or analogue of the phenothiazinecompound, wherein the phenothiazine compound is selected from the groupconsisting of ACV-1-235 (ACV); JJM-II-221E (221E); and DB1026(PerSucc)having the following structures:

Paragraph 12, a method of treating a subject with a ribosomal disorderor ribosomopathy, comprising administering an effective amount aphenothiazine compound, or a derivative or analogue of the phenothiazinecompound, and wherein the phenothiazine compound is selected from thegroup consisting of DB-4-083 (083); DB-4-084 (084); DB-4-088-2 (088-2);DB-4-088-3 (088-3); DB-4-086 (086); DB-4-087-2 (087-2); DB-4-087-3(087-3); DB-4-089 (089) having the following structures:

Paragraph 13, a method of treating a subject with a ribosomal disorderor ribosomopathy, comprising administering an effective amount of acomposition comprising a phenothiazine compound, or a derivative oranalogue of the phenothiazine compound, wherein the phenothiazinecompound is a compound of Formula (I):

wherein:

-   -   X is O or S;    -   R¹ is H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyls, acyl,        aryl, heteroaryl, alkylheteroaryl or alkylaryl;    -   each R is independently H, halo, alkyl, alkyl, alkenyl, alkynyl,        haloalkyl, CN, OH, NH₂, alkylamino, dialkylamino, CO₂H, acyl,        SH, thioalkoxy, SO₂H, or SO₃H; and    -   isomers and pharmaceutically acceptable salts thereof.

Paragraph 14, a method of treating a subject with a ribosomal disorderor ribosomopathy, comprising administering an effective amount of acomposition comprising a a phenothiazine compound, or a derivative oranalogue of the phenothiazine compound, wherein the phenothiazinecompound is a compound of Formula (II):

wherein:

-   -   each R²¹ is independently selected from the group consisting of        H, halo, alkyl, haloalkyl, CN, OH, NH₂, alkylamino,        dialkylamino, CO₂H, acyl, SH, thioalkoxy, SO₂H, and SO₃H;    -   isomers and pharmaceutically acceptable salts thereof.

Paragraph 15, method of treating a subject with a ribosomal disorder orribosomopathy, comprising administering an effective amount acomposition comprising a phenothiazine compound, or a derivative oranalogue of the phenothiazine compound, wherein the phenothiazinecompound is a compound of structure:

Paragraph 16, a method of treating a subject with a ribosomal disorderor ribosomopathy, comprising administering an effective amount of the isa calcium channel blocker BAPTA-AM or derivative or analogue thereof tothe subject to decrease active p53 in at least one of CD34+ cellswherein BAPTA-AM has the following structure:

Paragraph 17, the method of any of paragraphs 1-16, wherein the subjectwith a ribosomal disorder has Diamond Blackfan Anemia (DBA) or inheritederythroblastopenia.

Paragraph 18, the method of paragraph 17, wherein the subject has DBA1,DBA2, DBA3, DBA4, DBA5, DBA6, DBA7, or DBA8.

Paragraph 19, the method of any of paragraphs 1-16, wherein the subjecthas a mutation in ribosomal protein 19 (RPS19).

Paragraph 20, the method of any of paragraphs 1-16, wherein the subjecthas a mutation in ribosomal protein selected from RPS7, RPS10, RPS19,RPS24, PRS26, RPS17, PRS27L RPS29. RPL35A, PRLS and PPL11.

Paragraph 21, the method of any of paragraphs 1-16, wherein the subjecthas a mutation in a ribosomal protein selected from the group consistingof: rPL2A, rPL2B, rPL3, rpL4A, rPL4B, rPL7A, rPL7B, rPL10, rPL11,rPL16A, rPL17A, rPL17B, rPL18A, rPL18B, Rp119A, rPL19, rPL25, rPL29,rpL31A, rpL31B, rPL36A, rPL40A, rPS1A, rPS6A, rPS6B, rPS14A, rPS15,rPS19, rPS23B, rPS25A, rPS26B, rPS29, rPS29B and rPS31.

Paragraph 22, the method of any of paragraphs 1-16, wherein the subjectis administered another therapeutic agent to treat the ribosomal proteindefect, selected from the group consisting of: corticosteroids, bloodtransfusions.

Paragraph 23, the method of any of paragraphs 1-16, wherein theeffective amount increases the number of CD71+ erythroid cells in thesubject.

Paragraph 24, the method of any of paragraphs 1-16, wherein theeffective amount increases hemoglobin levels in the subject.

Paragraph 25, the method of any of paragraphs 1-16, wherein the subjecthas a symptom of macrocytic anemia or craniofacial abnormalities.

Paragraph 26, the method of any of paragraphs 1-16, wherein theribosomopathy is myelodysplasia.

Paragraph 27, the method of any of paragraphs 1-16, wherein themyelodysplasia is 5q-myelodysplasia.

Paragraph 28, the method of any of paragraphs 1-16, wherein the subjecthas a mutation in Rps14 or decrease in Rps14 expression.

Paragraph 29, the method of any of paragraphs 1-16, wherein subject hasa symptom of dysplastic bone marrow.

Paragraph 30, the method of any of paragraphs 1-16, wherein theribosomopathy is Shwachman-Diamond syndrome.

Paragraph 31, the method of any of paragraphs 1-16, wherein the subjecthas a mutation in Sbds.

Paragraph 32, the method of any of paragraphs 1-16, wherein subject hasa symptom selected from: pancreatic insufficiency, bone marrowdysfunction, skeletal deformities.

Paragraph 33, the method of any of paragraphs 1-16, wherein theribosomopathy is Treacher Collins Syndrome.

Paragraph 34, the method of any of paragraphs 1-16, wherein the subjecthas a mutation in TCOF1 (nucleolar).

Paragraph 35, the method of any of paragraphs 1-16, wherein the subjecthas a symptom of craniofacial deformities.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose of skill in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents and otherpublications identified are expressly incorporated herein by referencefor the purpose of describing and disclosing, for example, themethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents. All references, patents and publications citedherein are incorporated by reference in their entirety.

EXAMPLES

Although any known methods, devices, and materials may be used in thepractice or testing of the invention, the methods, devices, andmaterials in this regard are described herein.

The examples presented herein relate to methods and compositionscomprising at least one inhibitor as disclosed herein for treatment of aribosomal disorder or ribosomapathy, for example, but not limited toDBA. Throughout this application, various publications are referenced.The disclosures of all of the publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains. The followingexamples are not intended to limit the scope of the claims to theinvention, but are rather intended to be exemplary of certainembodiments. Any variations in the exemplified methods which occur tothe skilled artisan are intended to fall within the scope of the presentinvention.

Materials and Methods

Embryo Manipulation and Chemical Treatment

Fish were maintained under approved laboratory conditions. Studies wereperformed on AB wildtype strains and hi2903, an insertional mutant inthe first intron of ribosomal protein S29 (rps29). Embryos weresubjected to chemicals diluted in E3. For screening, chemicals from ICCBBiomol Known Bioactive, Sigma, and Lopac libraries were tested at 1:300dilutions from library stock. Compounds were tested in two independentexperiments of 20 embryos each, so approximately 10 mutant embryos werescored per chemical. Compounds were diluted in DMSO or water and testedin doses from 5-50 μg/mL

In Situ Hybridization and Benzidine Staining

Whole-mount in situ hybridization (ISH) was performed as described(Thisse and Thisse, 2008). Antisense probes were synthesized fromdigested plasmid. O-Dianisidine was performed as described previously(Paffett-Lugassy and Zon, 2005).

Cell Culture and Infection

A549 and CD34⁺ cells were infected with previously characterizedlentiviral shRNA targeting RPS19 (Dutt et al., 2011). Unless otherwisenoted, drugs were added one day post infection, and cells were collectedfor analysis 3-6 days post infection.

Flow Cytometry and Immunofluorescence

For flow cytometry based measurement of protein levels, cells were fixedin 2% paraformaldehyde for 15 minutes at 37° C., and methanol was addedfor overnight incubation at 4° C. Cells were incubated for one hour in1:100 diluted p21 primary antibody (Cell Signaling 12D1) followed by onehour in conjugated secondary antibody and 1:50 diluted p53-conjugatedantibody (Cell Signaling 1C12). Immunofluorescence staining wasperformed as previously described (Dutt et al., 2011).

Example 1

Ribosomal protein mutations are common in patients with Diamond Blackfananemia (DBA), who have red cell aplasia and craniofacial abnormalities.The inventors have previously characterized a zebrafish mutant in rps29,a ribosomal protein in the small subunit. Rps29^(−/−) embryos havemorphological defects in the head, as well as decreased hematopoieticstem cells, hemoglobin, and staining of endothelial markers. Consistentwith other models of DBA, knockdown of p53 near completely rescues therps29 mutant phenotype. To identify chemicals that could rescue therps29 mutant phenotype, the inventors performed an in vivo chemicalscreen. Inhibitors were found to rescue morphological, endothelial, andhemoglobin phenotypes.

Zebrafish RPS29 mutants have p53-Dependent Hematopoietic Phenotypes

The zebrafish work has focused on the rps29 mutant (Amsterdam et al.,2004). The inventors have previously reported that Rps29 mutant embryosinitially have hematopoietic and endothelial defects (Burns et al.,2009). Rps29^(−/−) embryos have a defect in arterial specification,leading to decreased hematopoietic stem cells anddecreasedflk/expression in the intersegmental vessels at 24 hours postfertilization (hpf). Primitive erythropoiesis is specifically affected,as rps29^(−/−) embryos have less hemoglobin whereas primitivemyelopoiesis is unaffected. The rps29 mutant embryos have increasedapoptosis, as seen by changes in head morphology and TUNEL staining.Microarray analysis demonstrated an activation of p53 and its targets inthe mutant embryo. When a p53 mutation was crossed into the backgroundof the rps29 mutant, all of the hematopoietic and apoptotic phenotypeswere rescued. Herein, the inventors demonstrate a critical role of p53activation in rps29 mutant phenotypes. This characterization of therps29 mutant and identification of a p53-dependent mechanism wasrecently published in the Journal of Experimental Hematology (Taylor etal., 2012, which is incorporated herein in its entirety by reference).

Chemical Screen Finds Specific Chk2 Inhibitors Rescue rps29^(−/−)Defects

A screen was performed to identify chemicals that could rescue theendothelial and morphological defects of the rps29′ mutant embryo (FIG.5). In particular, we assessed that ability of CaM dependent kinases torescue rps29^(−/−) mutant embryos. Surprsisngly, out of a large numberof CaM dependent kinases, only Chk2 inhibitors rescued well and thenthrough in vitro kinase screens (See FIG. 12 and FIG. 13) we thendetermined the Chk2 inhibitors CCT and III are effective throughinhibiton of RSK and p70s6k. Rps29^(+/−) fish were incrossed, andembryos were collected for treatment at bud stage (10 hpf). Embryos weretreated from bud to 23 hpf with compounds of known bioactivity. Afterbeing scored for rescue of head morphology, embryos were fixed at 24 hpffor in situ hybridization and montoring of Hb expression rescue.

The Chk2 Inhibitor CCT Rescues Hb.

Chk2 inhibitor III resues Hb:

Other naphthalenesulfonamidesare known to inhibit calmodulin, includingA-7 and W-5, and were previously demonstrated to also rescue thevasculature defect.

Example 2

Specific phenothiazine derivatives increase hemoglobin in rps 29−/−embryos.

A Benzidine Assay was performed. For the drug treatment, rps29+/− wereincrossed and embryos were collected and treated at 50% epiboly,approximately 5 hours post fertilization (hpf). Benzidine staining wasperformed at 40 hpf as described previously (Paffett-Lugassy and Zon,2005). FIG. 25 shows the dramatic rescue of Hb expression, hemoglobinrescue in rps29−/− zebrafish of the compounds ACV and PerSucc. Thereason for differences in Hb levels in the 0 control is that these weredifferent experiments and there is variability in Hb among differentclutches of fish.

Example 3

Novel phenothiazine derivatives improve erythroid defects in multiplemodels of DBA

We previously demonstrated that zebrafish embryos with mutations in therps29 gene (which encodes the ribosomal protein S29) model DBA (Taylor,A. M. et al.). A subset of patients with mutations in RPS29 exists, wepreviously established that RPS29 is a DBA-causative gene (Mirabello, L.et al.). Consistent with other animal models of ribosomal proteindysfunction, p53 knockdown in the rps29−/− zebrafish rescues theerythroid defect. To find novel compounds that can rescue the phenotypesof DBA, we performed an in vivo chemical screen in our zebrafish DBAmodel and found that calmodulin (CaM) inhibitors block p53 activity andrescue the erythroid defect. Applying CaM inhibitors to human CD34+cells deficient in RPS19 relieved the block to erythroiddifferentiation. Additionally, injecting the FDA-approved CaM inhibitor,trifluoperazine (TFP), into a DBA inducible mouse model (Jaako, P etal.) significantly increased red blood cell number and Hb levels andreduced p53 activity in the bone marrow. TFP belongs to a family of FDAapproved antipsychotics called phenothiazines. These drugs easily crossthe blood brain barrier (BBB) and their long-term use is associated withdyskinesia and extrapyramidal effects (Kennedy, P. F. et al.) makingthem inappropriate for use in children. To overcome this limitation, weused a llibrary of novel phenothiazine derivatives that will potentiallynot cross the BBB FIG. 33. First, we tested the compounds in rps29−/−zebrafish embryos to determine if any could improve the erythroid defectFIG. 34. We looked at Hb rescue in the zebrafish. A 24 well plate wasseeded with 20 embryos per well, and at 50% epiboly the embryos weretreated with drug. The embryos were then stained for hemoglobin (Hb) andfixed in 4% PFA. The Eight compounds were tested and three (boxed infigure: 088-2; 088-3; and 089)) were able to increase Hb at lowerconcentrations than the positive control phenothiazine (fluphenazine[Flu]) (FIG. 35).

It is well established that CNS-active small molecules may haveimmediate neurological effects (e.g. impaired locomotor activity(Giacomini, N. J. et al.; and Boehmler, W. et al.)) in zebrafish.Zebrafish are an excellent in vivo model to study BBB permeability sincethe function of their BBB was shown to be similar to humans (Jeong, J.Y. et al.). To test these compounds for BBB permeability we used a highthroughput behavioral testing assay for screening compounds that willaffect zebrafish swimming and thus suggest BBB permeation. This assayinvolves placing one 6 day post-fertilization (dpf) zebrafish into awell of a 96 well plate, adding drug to each well and recording themovement for 1-2 hours using automated analysis imaging software (Rihel,J. et al.). Initially testing this assay with commercial phenothiazineswe were able to statistically determine if a drug increases or decreasesmovement compared to DMSO treatment in the zebrafish larva (data notshown). This allows us to use behavior as a proxy for pervading the BBBin a high throughput manner. We tested four novel derivatives along witha commercial phenothiazine (Flu), and a chk2 inhibitor, as a control,that should not affect behavior. Out of the selected derivatives, threealtered behavior (083, 088-3; and 089) and 089 was even toxic at thehighest dose. While one drug, 088-2, did not affect the swimmingbehavior suggesting that it does not get into the brain of the zebrafish(FIGS. 36 to 39).

We next tested three derivatives in our primary human in vitrodifferentiation model of DBA. To do this we expanded CD34+ peripheralblood mononuclear cells in culture for 2 days and induced ribosomalprotein deficiency with a lentirvirus containing RPS19 shRNA (FIG. 40).After 3 days the cells are placed into erythroid differentiation mediawith or without the novel derivatives and cultured for 5 days. Erythroiddifferentiation is then assessed by flow cytometry analysis for thetransferrin receptor, CD71+. Results demonstrated that both 088-2 and089 increased the percentage of erythroid precursor cells compared toDMSO treated (FIG. 41). In addition, the novel derivatives increased theabsolute erythroid number compared to DMSO treated (FIG. 41). Theseresults demonstrate that the novel derivative 088-2 improves theerythroid defect in both rps29−/− zebrafish embryos and in a human invitro model of DBA. Unlike the parent phenothiazine compounds, 088-2does not cause behavior changes in zebrafish, suggesting that thecompound is not getting in the brain. A table summarizing the activityof the compounds is found in FIG. 43.

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What is claimed is: 1-36. (canceled)
 37. A phenothiazine compound ofFormula (I):

wherein: X is O or S; R¹ is H, alkyl, alkenyl, alkynyl, cyclyl,heterocyclyls, acyl, aryl, heteroaryl, alkylheteroaryl or alkylaryl;each R is independently H, halo, alkyl, alkyl, alkenyl, alkynyl,haloalkyl, CN, OH, NH₂, alkylamino, dialkylamino, CO₂H, acyl, SH,thioalkoxy, SO₂H, or SO₃H; and pharmaceutically acceptable saltsthereof.
 38. The phenothiazine compound of claim 37, wherein Xis S. 39.The phenothiazine compound of claim 38, wherein R¹ is a ₁-C₆ alkyl. 40.The phenothiazine compound of claim 39, wherein R¹ is isopropyl.
 41. Thephenothiazine compound of claim 40, wherein each R is independentlyselected from the group consisting of H, halo, C₁-C₆alkyl,C₁-C₆haloalkyl, OH, C₁-C₆alkoxy, NH₂, NO₂, C₁-C₆alkylamino,di(C₁-C₆alkyl)amino, CN or CO₂H.
 42. The phenothiazine compound of claim41, wherein each R independently is selected from the group consistingof H, CI, F, Br, methyl, trifluoromethyl, OH, NH₂, NO₂, CN or CO₂H. 43.The phenothiazine compound of claim 38, wherein R¹ is an optionallysubstituted alkylaryl.
 44. The phenothiazine compound of claim 43,wherein R¹ is C₁-C₆ alkylphenyl.
 45. The phenothiazine compound of claim43, wherein R¹ is benzyl.
 46. The phenothiazine compound of claim 45,wherein each R is independently selected from the group consisting of H,halo, C₁-C₆alkyl, C₁-C₆haloalkyl, OH, C₁-C₆alkoxy, NH₂, NO₂,C₁-C₆alkylamino, di(C₁-C₆alkyl)amino, CN or CO₂H.
 47. The phenothiazinecompound of claim 46, wherein each R independently is selected from thegroup consisting of H, CI, F, Br, methyl, trifluoromethyl, OH, NH₂, NO₂,CN or CO₂H.
 48. The phenothiazine compound of claim 1, whereinphenothiazine compound is

or a pharmaceutically acceptable salt thereof.
 49. A pharmaceuticalcomposition comprising the phenothiazine compound of claim
 48. 50. Aphenothiazine compound of Formula (II):

wherein: each R²¹ is independently selected from the group consisting ofH, halo, alkyl, haloalkyl, CN, OH, NH₂, alkylamino, dialkylamino, CO₂H,acyl, SH, thioalkoxy, SO₂H, and SO₃H; and pharmaceutically acceptablesalts thereof.
 51. The phenothiazine compound of claim 50, wherein is H,halo, C₁-C₆alkyl, C₁-C₆haloalkyl, OH, C₁-C₆alkoxy, NH₂, NO₂,C₁-C₆alkylamino, di(C₁-C₆alkyl)amino, CN or CO₂H.
 52. The phenothiazinecompound of claim 51, wherein each R²¹ independently is H, CI, F, Br,methyl, trifluoromethyl, OH, NH₂, NO₂, CN or CO₂H.
 53. A phenothiazinecompound selected from the group consisting of DB-4-083 (083); DB-4-084(084); DB-4-088-2 (088-2); DB-4-088-3 (088-3); DB-4-086 (086);DB-4-087-2 (087-2); DB-4-087-3 (087-3); and DB-4-089 (089), having thefollowing structures:

pharmaceutically acceptable salts thereof.